EFFECTS O? ENTRAPERITONEAL {NJECTIONS OF UREASE UPON PERFORMANCE AND HEMATOLOGY OF GROWENG SWINE l. lMMUNiIATION ii. TOXICWY Thai: for the Dogma of Ph. D. MiCHlGAN STATE UNWERSITY Evin T. Kamegay 1963 THESIS This is to certify that the thesis entitled Effects of lntraperitoneal Injections of Urease Upon Performance and Hematology of Growing Swine I. Immunization ll. Toxicity presented by Ervin T. Kornegay has been accepted towards fulfillment of the requirements for Ph.D degree in Animal Husbandry g2. 4.144;, V Major fiofessor Date {iii’mjf’u ’1 7‘1 "I 4 3 Michigan State University ABSTRACT EFFECTS OF INTRAPERITONEAL INJECTIONS OF UREASE UPON PERFORMANCE AND HEMATOLOGY OF GROHING SHINE I. IMMUNIZATION II. TOXICITY by Ervin T. Kornegay Six trials were conducted to study the effects of intraperitoneal injections of urease upon the performance and hematology of growing pigs. The first three trials which included 81 pigs were designed to study the effects of urease immunization upon the following criteria: feed intake and gain, feed efficiency, serum antiurease activity, plasma urea N and ammonia N, urine urea N and ammonia N, intestinal urease activity and ammonia N levels. Urease prepared frcm Jackbean meal was used in Trials I and III while Sigma urease type II powder was used in Trial II. Starting doses ranged from 0.1 to 10 Sumner units per pound of bodyweight with the size of subsequent doses determined by arithmetic progression. .A corn- soybean ration fortified with minerals and vitamins was fed in all trials in the_immuniaation study. The pigs immunized with urease exhibited significantly greater serum antiurease activity than did nondmmunized pigs. In the first two trials, the low levels of urease appeared to be more effective in stimulating antibody production than the high levels of urease, while all levels of urease were equally effective in Trial III. In Trials II and III, the greatest serum antiurease activity occurred after about 28 days, while in Trial I the greatest activity occurred after 72 days. Ervin T. Kornegay Gain and feed efficiency of immunized pigs were not improved in Trials II and III; however, there was some improvement in Trial I at the 10 unit level. Plasma urea N levels were unchanged between treatment groups in all trials. In general, ammonia N levels of plasma were unchanged; however, the 0.5 unit treatment group (low) in Trial III had signifi- cantly lower plasma ammonia levels at 56 and 73 days on experiment. Intestinal sections plus contents (from the caudal end of the ileum) taken in Trials I and III revealed that the urease immunized pigs had less urease activity than the control pigs, with the difference between treatment and control groups significant in Trial III (P«; 0.05). Sigma urease type II powder used in Trial II was found to have high hemagglu- tinative activity. Urease immunization appeared to have no effect on the utilization of fed urea by growing pigs. Higher levels of urea N and lower levels of ammonia N were observed in pigs fed rations with added urea than in pigs fed the control ration. Gain, feed intake, and feed efficiency were decreased at the 2.5 and 3.h percent urea levels. Three trials were conducted in the toxicity phase to study the effect of a large intraperitoneal injection of urease on growing pigs and its relationship to ammonia toxicity. Urease prepared from Jackbean meal by the author was used in Trials I and II and Sigma urease type II powder was used in Trial III. Plasma urea N levels were significantly decreased, and plasma ammonia N levels were significantly increased fol- lowing a large intraperitoneal injection of urease (50, 75 and 100 modi- fied Sumner units per pound of bodyweight). All pigs receiving 75 and 100 unit levels died. Ammonia N levels in pigs which died ranged from 2.0 to 3.0 milligrams per 100 milliliters of plasma. Serum urease activity and potassium levels were increased, and serum protein, sodium and calcium levels were unchanged in pigs given a large dose of urease Ervin T. Kornegay (Trials II and III). Liver urease activity and ammonia N levels of the urease inJected pigs were larger than for the control pigs though the differences were not statistically significant (Trial II). Urine am- monia N levels of treated pigs were larger than values for control pigs in Trials I and II while the opposite was true in Trial III. Urine urea N levels were decreased in Trial III and unchanged in Trials I and II. The electrophoretic components of serum protein in Trial III showed gamma globulin of urease injected pigs to be significantly greater than values for the control pigs. The other components of serum protein were not significantly different. Serum transaminase values determined in Trial III disclosed that glutamic-oxalacetic values were unchanged and that glutamic-pyruvic values were lower following a large urease in- Jection. There was an elevation of rectal temperatures of treated pigs in Trials I and II with no change in Trial III. Pigs in Trials I and II showed tetany while those in Trial III did not. Post-mortem examination revealed excess fluid in the peritoneal and pericardial cavities, con- gested and hemorrhagic lungs, and hyperemic mucous membranes. Effects of urease and hemagglutinin were confounded in Trial III which could explain the differences in results obtained in Trials I and II, and Trial III. EFFECTS OF INTRAPERITONEAL INJECTIONS 0F UREASE UPON PERFORMANCE AND HEMATOLDGY OF GROWING SWINE I. IMMUNIZATION II. TOXICITY BY Age”; 3/ h Ervin T. Kornegay A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Husbandry 1063 I ACKNOWIEDGMENTS The author is grateful to Dr. J. A. Hoefer, Dr. E. R. Miller and Dr. D. E. Ullrey for their helpful advice, able instruction and guidance throughout his graduate study, and for their critical reading of this manuscript. The writer also wishes to express his sincere appreciation .to other members of his guidance comittee, Dr. R. H. Luecke and Dr. E. P. Reineke, for their interest and assistance. Sincere gratitude is expressed to Dr. R. 8. Nelson, Dr. J. A. Roofer, Dr. 3. R. Miller and Dr. D. E. Ullrey for the materials and facilities which made this research possible and for the financial sup- port of Michigan State University through an ass istantship. The author wishes to thank Mr. M. 3. Newton and others at the swine far- for their assistance in the care and nnagement of the pigs, and Mrs. w. H. Vincent, Mr. c. L. Zutaut and Mr. G. V. Grossnn for their able assistance in biological determinations. Special acknowledgment is due the Backha- l'oundation for support of this study. The writer wishes to acknowledge his gratitude and indebtedness to his parents for their unwavering assistance and encouragement throughout his educational career. To Junita, his wife, the author extends his thanks for proofreading and typing the manuscript and for her cooperation, encomgeaent , and sacrifices which helped make graduate study possible. Irvin T. Kornegay candidate for the degree of Doctor of Philosophy DISSERTATION: Effects of Intraperitoneal InJections of Urease Upon the Performnce and Bentology of Growing Swine I . Imunisat ion II. Toxicity mm 0’ 811mm: Main Area: Ania-s1 Husbandry (Aninsi nutrition) Minor Areas: Biochemistry, Pivsiology BIMCAL m: Born: lurch 16, 1931. Paison, North Carolina Undergraduate studies: I. C. State College, 1919-1953 Graduate studies: 1!. C. State College, 1958-1960 Hichigan State university, 1960-1963 mm: Officer United States Army, 1953-1955 Assistant Agricultural Agent, 1955-1958 maduate Assistant, I. C. State College, 1959-1960 Graduate Assistant, Michigan State University, 1960-1963 mm: American Society of Animal Science Gann- Sign Delta Society of Sign Xi iii I. II. III. INTRODUCTION REVIEW OF LITERATURE A. B. H. Ammonia Production urease Activity and Urea Hydrolysis TABLE OF CONTENTS 0 O O O O O O O O O O O O O O O O O O O O O O O O Urease Occurrence in Nature and Origin in Animals Comparison of Urease from Various Sources Isolation and PrOperties of urease Physiological Role of Urease . urease and Ammonia Toxicity . . urease Immunization . . . . . . EXPERIMENTAL PROCEDURES . . . . . . A. Immunization . . . . . . . . . 1. General . . 2. Trial I . . 3. Trial II. . he Trial III a 5. Urease . . a. b. c. C O O O O O O O O 0 e O 0 e O 0 Preparation . . . . . . Enzyme activity 0 C O O 0 O Hemagglutinative activity 6. Biological determinations . . a. be C. d. Po .1 f. Ammonia N . . . . . . . Serum antiurease activity Preparation of intestinal sections Intestinal ammonia and urease activity 0 O O O O 0 Serum antihemagglutinative activity . . Hematocrit, hemoglobin and urea N . . 1v 0 O C O O . (DWWW ll l2 l5 17 21 V. VI. VII. VIII. TABLE OF CONTENTS (CONTINUED) B e TOXICIty e a o e e e e 0 s 0 e a 1. General . . . . . . . . . . . 2. Biological determinations . . a. b. c. d. e. f. g. Glucose O O O O O O O O 0 Serum protein . . . . . . ElectrOphoresis . . . . Serum sodium and potassium Total nitrogen . . . . . Serum calcium . . . . . . Serum glutamic-oxalacetic transaminase . . . . . . RESULTS AND DISCUSSION . . . . . . . A. Immunization . . . . . . . . . . 1. hial I O O O O C O O O O O O 2. ”1.1 II 0 O O O O O O O O O 3. fiial III 0 O O O O O O O O O B. Toxicity . . . . . . . . . . . . ls Trial 1 s s e e e e s e s s e 2e Trial II a e e e e e e s e s 30 Trial III e e e e e e e s e e SUMMARY . conchIONS O O 0 O O O O O O O O O O BIBLIOGRAPHY . . . . . . . . . . . . APPENDIX and C O O O O 0 glutamic-pyruvic O O O 0 O O O O 0 O O O O O O O O O O O O O O O O Page LIST OF TABLES Table 1. 2. 10. ll. Immunization study. Composition of rations . . . . . . . . Trial I. Feed consumption, average daily gain and feed efficiency of urease immunized and nonimmunized growing pigs 0 I I O I O O O O O O O O O O O O O O O O I O O 0 Trial I. Plasma urea N and ammonia N levels, and serum antiurease activity of urease immunized and nonimmunized growing pigs . . . . . . . . . . . . . . . . . . . . . . Trial I. Effect of initial and secondary injections of urease upon plasma urea N and ammonia N levels of growing pigs . . . . . . . . . . . . . . . . . . . . . . Trial I. Effect of a secondary injection of urease on urine flow, urea N, and ammonia N of urease sensitized growing pigs . . . . . . . . . . . . . . . . . . . . . . Trial I. urease activity and ammonia N levels of intesti- nal sections plus contents from urease immunized and nonimmunized pigs . . . . . . . . . . . . . . . . . . Trial II. Peed consumption, average daily gain and feed efficiency of urease immunized and nonimmunized growing. pigs 0 O O O O O O O O 0 I O O O O O O 0 e I O O O O 0 e Trial II. Plasma urea N and ammonia N levels and serum antiurease activity of urease immunized and noninmunized “Wing pigs 0 O 0 O O O O O O O O O O O O O O O O C O 0 Trial II. Peed consumption, average daily gain and feed efficiency of urease immunized and nonimmunized growing pigs fed urea to supply part of the protein requirement. Trial II. Plasma urea N and ammonia N levels of urease immunized and nonimmunized growing pigs fed urea to supply part of the protein requirement . . . . . . . . . Trial III. Feed consumption, average daily gain and feed efficiency of urease immunized and nonimmunized growing pigs 0 O O O s e O O O O O O O 0 e O I O O O O O 0 O O 0 Trial III. Plasma urea N and ammonia N levels and serum antiurease activity of urease immunized and nonimmunized amine pig. 0 I O O O I O O O O O O O O O O O O O O O 0 vi an ’45 1+7 A8 1‘9 50 51 5h 56 58 59 Table 13. 11.. 15. 16. 17. 18. 19. 20. LIST OF TABIES (CONTINUED) ii Trial III. Feed consumption, average daily gain, feed efficiency, plasma urea N and plasma ammonia N levels of urease immunized and nonimmunized pigs fed 3.h percent urea to supply part of the crude protein requirement . . . . . . . . . . . . . . . . . . . . . . 61 Trial III. Urease activity and ammonia N levels of intestinal sections plus contents from urease immunized and nonimmunized pigs . . . . . . . . . . . . . . . . . 62 Trial II. Serum urease activity, protein, potassium and sodium of growing pigs injected intraperitoneally with 50 and 100 80 of urease per pound of body weight . . . . 66 Trial II. Urease activity and ammonia N levels of liver; and urine flow, urea N, and ammonia N level of growing pigs injected intraperitoneally with 50 and 100 SD of urease per pound of body weight . . . . . . . . . . . . 67 Trial III. Effect of an intraperitoneal injection of 75 SU of urease per pound of body weight upon hematocrit, hemoglobin, blood glucose and rectal temperature of growing pigs . . . . . . . . . . . . . . . . . . . . . . 71 Trial III. Effect of an intraperitoneal injection of 75 $0 of urease per pound of body weight upon serum urease activity, electrolytes, and transaminase activity in growing pigs . . . . . . . . . . . . . . . . . . . . . . 73 Trial III. Effect of an intraperitoneal injection of 75 SU of urease per pound of body weight upon electro- phoretic patterns of serum . . . . . . . . . . . . . . . 7h Trial III. Urine flow, total N, urea N, ammonia N'and difference N of growing pigs given high urease inJCCtioMeessoeeeeeeesseseesesss 75 vii LIST OF FIGURES Figure l. ElectrOphoretic pattern of Sigma urease type II and ureasepreparedbytheauthor.............. 2. Trial 1. Effect of an intraperitoneal injection of 100 $0 of urease per pound of body weight on plasma urea and ammonia of a growing pig . . . . . . . . . . . . . . . . 3. Trial II. Effect of intraperitoneal injections of 50 and l00 50 of urease per pound of body weight on plasma urea andammoniaofgrowingpigs............... h. Trial III. Effect of an intraperitoneal injection of 75 SU of urease per pound of body weight on plasma urea and monuorgmingpigseeeeeseeeeeeeeees viii in? 53 53 65 69 Table 1. 10. ll. 13. LIST OF.APPENDIX TABLES Trial 1. Age, sex, initial weight, final weight, and average daily gain of growing pigs in immunization study (pounds) . . . . . . . . . . . . . . . . . . . . Trial I. Plasma urea N levels in immunization study (MO/1m ml.) 0 O O O O O O I O O O O O O O O O O O O 0 Trial 1. Plasma ammonia N levels in immunization study (meat/1w ml.) 0 O O O O O O O O O O O O O O O O O O 0 0 Trial I. Serum antiurease activity in immunization study (units/ml. x 103) O O O O O O O O O O I O O O O O O O 0 Trial I. Plasma urea N and ammonia N levels of growing pigs following initial and secondary injections of urease O O O O O O O O O O O O C O O C C O O O O O O O 0 urine flow, urea N and ammonia N of growing pigs given a secondary injection of urease in immunization study . . Trial 1. urease activity and ammonia N levels of intesti- nal sections plus contents from pigs in immunization 8 tudy e s e e s e s a e e e s s s e 0 s e e e O s e 0 Trial II. Age, sex, initial weight, final weight, and average daily gain of growing pigs in immunization Study (pounds) 0 e e e e o e e e O a e e e e e e e e e 0 Trial II. Plasma urea N levels in immunization study (mg./100 m1.) . . . . . . . . . . . . . . . . . . . . . Trial II. Plasma ammonia N levels in immunization study (meg./100m1.)..................... Trial II. Serum agtiurease activity in immunization study (unit8'/m10 x 10 ) O O O O O O O O O O O O O O O O O O 0 Trial II. Average daily gain of immunized and nonimmunized growing pigs fed urea to supply part of the crude protein requirements (pounds) . . . . . . . . . . . . . . . . . Trial II. Plasma urea N levels of immunized and nonimmunized growing pigs fed urea to supply part of the crude protein requirements (mg./100 ml.) . . . . . . . . . . . . . . . ix ii“ 9h 95 96 97 100 101 103 10h 105 106 107 LIST 0? APPENDIX TABLES (CONTINUED) 6? Tahle __§_ 1h» Trial II. Ammonia N levels of immunized and nonimmunized growing pigs fed urea to supply part of the crude protein requirement (mcg./100 ml.) . . . . . . . . . . . . . . . 108 15. Trial III. Sex, initial weight, final weight, and average daily gain of growing pigs in immunization study . . . . 109 16. Trial III. Plasma urea N levels in immunization study (m8. /lm ml.) 0 C O O O C O O C O O C O O O O O C O O C 110 17. Trial III. Plasma ammonia N levels in immunization Stady (meg‘/100 m.) 0 O O I O O O O O O O O I O O O O O 111 18. Trial III. Serum antiurgase activity in immunization BtUdy (units /ml 0 x 10 ) O O O C O O O O O O O O O O O O 1.12 19. Trial III. Average daily gain, plasma urea N and ammonia N levels of immunized and nonimmunized pigs fed 3.h percent urea to supply part of the crude protein requirement . . . . . . . . . . . . . . . . . . . . . . 113 20. Trial III. urease activity and ammonia N levels or intesti nal sections plus contents from pigs in immunization atUdy 0 O I O O O O O O O O O O O O O O O O O O O O O O 115 21. Trial 1. Plasma and urine urea N and ammonia N levels in tox 1c ity stm O 0 O O O O C O O C O O 0 O O O O O O 0 O 116 22. Trial II. Plasma urea and ammonia N levels in toxicity Study 0 O O O O O O O O O O O O O O O O O O O O O O O O 117 23. Trial II. Serum urease activity, potassium, sodium and protein in toxicity study . . . . . . . . . . . . . . . 118 2h. Trial II. urease activity and ammonia N level of liver; and urinary flow, urea N, and ammonia N level in tox1C1ty Bt‘ny C O O C C O O O O O O O O O O O O O O O O 119 25. Trial III. urea N and ammonia N levels in toxicity study . 120 26. Trial III. Hematocrit and hemoglobin levels in toxicity Btw O C O O O O O C O O O O C O O O C O O C O C C O C 121 2?. Trial III. Blood glucose and temperature in toxicity Study a a a o a O O O O O O O O O O O O O O O 0 O O O O 122 X Table LIST 01“ APPENDIX TABLES (CONTINUED) 28. Trial III. Serum urease activity and potassium level in 29. 30. 31. 32. tOXicj-ty .tw O O O O O O O O O O O O O O O O O 0 O O 0 Trial III. Sodium and calcium levels in toxicity study . . Trial III. Serum glutamic-pyruvic and glutamic-oxalacetic transaminase in toxicity study . . . . . . . . . . . . . Trial III. Serum electrophoretic pattern in toxicity study 0 O O O O C O O I O O O O O I 0 O O O O O C O O a Trial III. Urine flow, total N, urea N, ammonia N and difference N in toxicity study . . . . . . . . . . . . . xi £253. 123 12% 125 126 128 I. INTRODUCTION Antimicrdbial agents have been effective in improving the perform~ ance of animals under a variety of environmental conditions. Most investigators agree that these agents alter the microflora of the gastro- intestinal tract. Some believe that a toxic material produced by the microflora is eliminated or its production is inhibited by low level antimicrobial agents in the diet. Frost 53 51, (1955), Jukes (1957) and Visek (1962) have reviewed this subJect. Ammonia has been suggested as one of the toxins, perhaps the most important one, whose production is inhibited by low levels of antimicro- bial agents in the diet (Francois and Michel, 1955; Helnykawycz and Johansson, 1955; Sherlock, 1958; Dang and Visek, 1960; Visek, 1962). The gastrointestinal tract has been shown to be the major site of ammonia production in the animal (Dintzis and Hastings, 1953; Lawrence gt_gl., 1958). Studies with isotOpically labeled urea and antibacterial agents have demonstrated that urea is hydrolyzed in the gastrointestinal tract by urease, the only enzyme known to break down urea to 002 and ammonia (Dintzis and Hastings, 1953; Kornberg gt 3;” l95ha,b; Visek, 1962). Urease has been reported to occur in over 200 species of bacteria as well as in higher plants and animals (Sumner and Somers, 1953). Raw- ever, the exact origin of urease in the animal body is not agreed upon by all investigators. Most investigators believe that gastrointestinal urease is of bacterial origin while a few believe that tissues of higher -1- .ll 0) II All I (III III. ’II‘ (III I {ill I | LIIII ‘ll [till will! III!) In! - 2 - animals also produce this enzyme. Parenteral injections of high levels of Jackbean urease have been shown to be toxic to a number of laboratory animals (Carnot 33 21., 1921; Kirk and Sumner, 1931; Tauber and Kleiner, 1931; Bandford, 1961; Dang and ‘Visek, 1963). Death.in.urease poisoning, however, is believed to be caused by the ammonia released. The antigenicity of urease was shown soon after its crystallization (Kirk and Sumner, 1931). Recently, Visek and coworkers have demonstrated improved growth performance of rats and chicks immunized with Jackbean urease (Dang and Visek, 1960; Visek and Dang, l960a,b; flarbers gt_gl., 1962, 1963b). Since no work.with urease has been reported in swine, this study was initiated to determine: 1) the effects of Jackhean urease immuniza- tion upon the performance and hematology of growing pigs; 2) the effects of a large intraperitoneal injection of Jackbean urease on growing pigs and its relationship to ammonia toxicity. II. REVIEH 0? LITERATURE ~ A, Ammonia Production Studies with liver disease, hepatectomy, and gastrointestinal tract removal have confirmed conclusions drawn from experiments with antibac- terial agents that the gastrointestinal tract is the major site of ammonia production in the animal body. Moore 23.31. (l9h6) reported accelerated growth in chicks (free of clinical disease) fed a purified diet containing succinylsulphathiazole and streptomycin. These workers postulated that gastrointestinal bac- teria could have an inhibitory effect upon growth without causing clini- cal disease. They suggested further that these dietary supplements were inhibiting microflora which produced toxic substances or made vita- mins unavailable. However, they pointed out that the possible systemic action of these antibacterial agents could not be overlooked. Since these early observations, extensive literature on the use of antimicrdbial agents has accumulated and many studies have been con- ducted in an attempt to elucidate the mechanism involved. A majority of investigators agree that these agents alter the microflora of the gastro- intestinal tract. The removal or inhibition of a toxic material pro- duced by the microflora is often mentioned as an explanation for the improved growth perfhrmance when antimicrobial agents are fed at low levels. Ammonia has been suggested as one of the most important toxins (Francois and Michel, 1955; Helnyknowycz and Johansson, 1955; Michel and Francois, 1955; Sherlock, 1958; Bang and Visek, 1960; Visek, 1962). - 3 - - h - Nancki gt_gl. in 1896 demonstrated that the portal blood ammonia concentration was much higher than that of the peripheral blood. Polin and Denis (1912) postulated that the higher portal blood ammonia concen- tration was the result of ammonia production by intestinal bacteria acting on nitrogenous substrates. This view was supported later by studies of Phear and Ruebner (1956) and by studies of the effects of antibiotics on ammonia production in the gut (Dintzis and Hastings, 1953; Silen gt_gl,, 1955). Bessman (1959) in a review article states that there are three sources of ammonia in the organism: gut, kidney and muscle. In the gut, ammonia comes from the hydrolysis of glutamine which is formed from pro- tein by hydrolytic enzymes and from the hydrolysis of urea by urease. The renal venous outflow contains more ammonia than the arterial inflow. This probably results from the back diffusion of ammonia from the tubules of the kidney during secretion of ammonia in the urine. urinary ammonia does not come from blood ammonia, but from the hydrolysis of glutamine by glutamimse to ammonia and glutamic acid in the kidney. Other major sources of urinary ammonia are derived from the action of D- and Leanino acid oxidases on the corresponding amino acids. In- creased amounts of ammonia appear in the venous blood following muscle contraction. It is now established that the removal of ammonia from the blood is defective in the presence of liver disease (Singh st 31., l95h) and that intestinal bacteriostasis diminishes the toxic symptoms of patients with impending hepatic failure. FalcOn and Iisher (1959) showed that neomycin therapy reduced blood ammonia levels in hepatic coma. Iawrence gt_gl. (1958) reported that the severity of the rise in blood - 5 - ammonia of a dog following hepatectomy was less when the animal was treated previously with neomycin. Bollman and Mann (1930) and Lawrence gt_al, (1958) noted a progres- sive r'ise in blood ammonia after total hepatectomy in dogs and demon- strated that removal of the gastrointestinal tract prevented this rise to a large degree. These findings re-emphasize the importance of the gastrointestinal tract in the production of ammonia. B. Urease Activity and Urea Hydrolysis For many years, urea was considered an end product of amino acid metabolism in mammals and was thought to be metabolically inert in simple-stomached animals. It is now believed that urea hydrolysis lg 3332 is an enzymatic process and that urease is the only known enzyme to decompose urea to carbon dioxide and ammonia (Dintzis and Hastings, 1953; XOrnberg gt 21., l95ha,b; Kornberg and Davies, 1955; Visek, 1962). Luck (l92hb) in his early studies of the presence of urease in the gastric mucosa of a number of animals suggested that urea was broken down to carbon dioxide and ammonia. Reports by Schoenheimer (l9h2) and Bloch (19h6) using urea-N15 indicated that only a very small portion of the nitrogen of ingested urea was transformed to ammonia and protein nitrogen, while most of the labeled urea was excreted as such. Recent studies, however, in several species of animals have re- vealed that isotOpically labeled urea is hydrolyzed in the alimentary tract even when injected parenterally. Leifer st_gl, (l9h8) found that 21 percent of urea-Cl“ injected intraperitoneally into mice appeared in 11‘0 in h8 hours. Kornberg and Davies (1952) re- 2 ported that cats injected intravenously with urea-Cl“ the expiratory air as C expired 1 to 2 per- cent of the urea as Clho2 in h to 5 hours. when the cats were injected - 5 - with urea-N15 subcutaneously, they found that approximately 5 percent of the urea was broken down after ho hours. Hastings 33 21, (1950) using urea-01h confirmed that urea was broken down to carbon dioxide and ammonia in the animal body. Gastrectomized mice excreted urea carbon as carbon dioxide at about one-half the rate of normal mice. Removal of the stomach and intestinal tract practically eliminated the excretion of urea carbon as carbon dioxide. Levenson gt 21. (1959) using germ-free and conventional rats com- pared the utilization of urea-c1h administered by subcutaneous injec- tion and by stomach tube. The conventional rats expired 2 percent of the dosed urea-C1h as CIhOé in 6 hours. The germ.free rats expired only 1/1oo as much in the same period of time. The pattern of urea metabolism was similar when urea was given either subcutaneously or by mouth. The small fraction of C11‘ expired by the germ-free rats indicated that some urea was being broken down. They pointed out, however, that urea is spontaneously hydrolyzed 2.9. m at pH 7 and 37°C. nus would account for the breakdown of urea in the germ-free rats. Studies of the rate of urea decomposition in cats (Hyatt and Kornberg, 1952; Kornberg 33 21,, l95hb) showed that the factor must be enzymatic in nature since the observed rate of breakdown was 10,000 times faster than the rate of uncatalyzed spontaneous decomposition. Dintzis and Hhstings (1953) reported urease activity to be five times greater in the last 5 centimeters of the small intestine and in the colon than in the stomach. Feces were found to have a high ureolytic activity. Studies by Liu :3 51. (1955) in pigs, Rose and Dekker (1956) in rats and Halser and Hodenlos (1959) in humans showed by using - 7 - isotopically labeled urea in the diet that a small but definite amount of the ingested urea could be found later incorporated into body protein. Rose and Dekker (1956) fed one treatment group of rats a diet in which the protein was made up of the essential amino acids only in the minimum required amounts. A second group of rats received a casein diet. Both diets contained 1.23 percent urea-N15. The distribution of the isotOpe in the excreta and carcass protein indicated an extensive utilization of the urea nitrogen by the rats on the amino acid diet, but not by those on the casein diet. cystine, glutamic acid and aspartic acid obtained from the former group had a high body concentration of the N15. On the other hand, the dietary essential amino acid histidine had an extremely low level of N15. Visek 2?. 11. (1959) showed that the addition of 100 ppm. of either chlortetracycline, penicillin or arsenilic acid to a casein diet de- creased $3.!igg_hydrolysis of urea-C1h by rats and reduced in 11352. ureolytic activity of gastrointestinal contents. A recent study by Harbers gt_gl. (1963a) showed that barbituric acid and a combination of barbituric acid and chlortetracycline, but not chlortetracycline alone, fed to chicks were effective in lowering the ureolytic activity and ammonia level of the small intestine. Dang and Visek (1963) reported that rats and mice immunized.with jackbean urease metabolized less urea-c1h 12,1132, Gastrointestinal urease activity of the inunised animals was reduced 60 percent from controls and the ammonia concentration of gastrointestinal contents was reduced 30 percent. lebers g§_gl. (l963b) showed that both jackbean urease immunization and increased vitamin A levels suppressed urease activity in the gastrointestinal tract. - 3 - C. Urease Occurrence in Nature and Origin in Animls Musculus in 1876 was the first to report experiments with the en- zyme, urease (Varner, 1960). He obtained urease by filtering bacteria on filter paper, drying the paper and using the dry paper, impregnated with an acid-base indicator, to test for urea. Miquel (1890) observed urease in many species of microorpnisms. Urease has now been reported to occur in over 200 species of bacteria as well as in higher plants and aninmls (Sumner and Sorters, 1953. The authors did not indicate whether the bacteria were aerobic or anaerobic). Huet and Aladame (1952) assayed over 200 anaerobic bacteria and found only four species which possessed urease. These and other workers (Gibbons and Doetsch, 1959) concluded that urease was sparse among anaerobic forms of bacteria. The richest plant source knowu is the jackbean (Canavalia ensiformis, Canavali obtusifolia) which contains 0.15 percent urease (on a dry weight basis). This is approxintely 16 times the urease con- tent of the soybean (mteer and fireball, 1916). The richest known source of urease is Bacillus pgsteurii which contains up to one percent of its dry weight as urease (Iarson and Kallio, 1951+). Urease has also been reported in the liver of two species of mollusks , in one species of worm, in a crustacean, in the horseshoe crab and in the larvae of the blowfly (Varner, 1960). The presence of urease in gastric mucosa was first demonstrated by Luck (192%) who found that suspensions of dog liver, when mixed with suspensions of dog gastric mucosa, liberated considerably more ammonia than either suspension alone, and that as a result the urea content of the mixture decreased to zero (Luck, 192%). When urea solutions were incubated with the gastric mucosal suspension, stoichiometric amounts - 9 - of ammonia were formed. Luck, therefore, concluded that urease was present in the gastric mucosa. EXperiments‘gg‘zitrg have shown that gastric urease occurs in a large variety of animals. urease is reported to occur in the stomach of men (Cardin, 1933; FitzGerald and Murpw, 19h9; Glick, 19h9), cats (Davies and Kornberg, 1950; ritzseraid and Murphy, 1950), chicks (Barbers 33 31.496312), nice (ritzseraid and Murpm', 1950 ; Davies and Kornberg, 1951), sheep, bullocks and goats (Luck, l92hb) and in two varieties of frogs (Glick gt_gl., 1950; Korff SE 31,, 1951). In contrast, the pres- ence of urease in the stomach of rabbits, rats, and pigs has been dis- puted (Luck, l92hb; Linderstrom-Lang and Ohlsen, 1936; Neil, l9hh; PitzGerald, 19h6; ritzoeraid and Murphy, 1950; Chao and Tarver, 1953} However, Cheosakul gt 21. (1958), Visek gt a}: (1959), Deng and Visek (1963) and Harbers gt_gl, (1963b) have reported urease activity of the gastrointestinal contents of rats. Much of the evidence that urea is hydrolyzed by urease in simple- stomsched animals has been reviewed recently by Visek gt_gl. (1959) and Visek (1962). The exact origin of urease in the animal body is not agreed upon by all workers. Visek (1962) points out that the prepon- derance of evidence favors Kornberg and Davies' (1955) conc1usion that urease is not endogenously produced by tissues of warm blooded species. Kornberg and Davies' (1955) conclusion was based mainly upon the inhibitory effect that antibiotics have on urease activity. Experiments by Dintzis and Hastings (1953). and Kornberg 2?. 31. (195nm) showed that ig_!igg'urea hydrolysis may be completely suppressed by high dietary levels of penicillin, oxytetracycline, and sulfaguanidine. Experiments with mice and cats showed that antibiotics indirectly affect urease activity by inhibiting the activity and growth of - 10 - intestinal microorpnisms. Dintzis and Hastings (1953) with mice and Kornberg gt a}: (195ha) with cats showed that a mixture of penicillin, terramvcin and sulfaguanidine (PTS) did not inhibit the urease activity of mucosal suspensions lg _v_i_‘_t_r_q and that no urease inhibitor was formed in the tissue as a result of PTS treatment (Kornberg and Davies, 1955). Also, studies with cats subjected to partial gastrectow before and after treatment with PTS (Kornberg 33 51., 195k) showed that PTS did not directly inhibit urease in 11152, but exerted its effect by inhibiting or eliminating urease-containing bacteria. It was also noted that after 3 days of treatment with PTS, the stomach contained urease despite a total absence of urea-splitting microorganisms, but that after 5 days of treatment both bacteria and urease activity ind disappeared (Kornberg g 51. , 195'“). The authors interpreted this to mean that the urease was of bacterial origin, that it had been deposited within the mucosa at a site protected from inactivation by acid and pepsin, and tint it was (after Pl‘S treatment) broken down in the noruml course of protein metabo- lism. The high dietary levels of PTS used by these workers reduced fecal bacterial counts from 106 to about 50 per milligram. However, fecal bacterial counts have not been consistently changed, either quantita- tively or qualitatively, in birds and Im-mls fed low levels of anti- bacterial agents for growth stimulation (Brande gt 2., 1953; Jukes, 1957) . The conclusion that gastrointestinal urease is of bacterial origin is strongly favored by the work of Levenson e__t_ 21. (1959) who obtained no evidence for enzymtic hydrolysis of urea in germ free rats. Halser and Hodenlos (1959) using both N15 and 01" labeled urea to study urea metabolism in man concluded that in man, as in animals, a - 11 - considerable fraction of synthesised urea is continuously being degraded by intestinal bacteria. The following lines of evidence were presented in support of their conclusion: 1) the estimated rate of urea produc- tion exceeded the measured rate of urea excretion by approximately 20 percent; 2) the recovery of labeled urea in the urine following intra- venous injections was incomplete; 3) ten percent of the excess N15 ap- peared in urinary ammonia and assures nitrogen; h) a significant frac- tion of the labeled urea molecule in plasma, following N15 injection was found to be newly synthesized; 5) the recovery of labeled urea in the urine became nearly complete following oral ingestion of neomycin. Conway st 21. (1959), however, believe that gastric urease is not entirely of bacterial origin. They base their conclusion chiefly on the following points: 1) the urease in the gastric mucosa is contained with- in the surface epithelial cells, which contain no bacteria; 2) the amount of urease in the animal is relatively large and would require such a large bacterial papulation to supply it that a bacterial origin for such urease appears untenable; 3) the fetus has gastric urease (cu-din, 1933). It is believed by Kornberg and Davies (1955) that gastric urease must be situated within the stomach wall at a site protected from.attack by acid.and pepsin. D. Comparison of Urease from Vhrious Sources Kirk and Sumner (1932) in a study of soybean and jackbean urease reported that the two were immunologically identical and that they were equally toxic at high levels. Nikoloff (quoted in Varner, 1960) made an extensive comparison of cat gastric urease and plant urease and con- concluded that there are two ureases in the gastric mucosa, both - 12 - different from plant urease in electrOphoretic and imunologic proper- ties. Conway g_t_ 91. (1959) stated: "Mouse gastric urease is found to have an acid Optimum, and its pH activity curve showa that it has a greater tolerance of acid conditions tlnn the urease of the jackbean or of the bacterium examined. This seems to indicate that yatric urease is not identical with vegetable or bacterial urease. " Seneca et- 11. (1962) recently reported the molecular weight of bacterial urease to be ‘$73,000 which is the same as reported by Sumner at 21. (1938) for jack- bean urease . E. Isolation and Pr0perties of Urease Urease was the first enzyme to be isolated in crystalline form (Sinner, 1926). The procedure develOped by Sumner (1926) which appears very simple is still in use today. However, Gorin e_t_ a_l_. (1962) state that their first attempts to prepare crystalline urease by the Sumner procedure were unsatisfactory and it became apparent that rigid control of all experimental conditions was necessary to obtain reproducible re- sults. They pointed out further that Summer's method did not afford a good recovery of the ensylmtic activity from the starting smterial, but that modifications effective in extracting more enzyme would also ex- tract more inactive nterial which interfered with the subsequent puri- fication. Handford (1961) states: ”Attempts to prepare crystalline urease from .hck Hean meal by the acetone extraction method of Sumner (1926) were unsuccessful." In Sumner's method, 100 grams of jackbean meal is stirred into 500 milliliters of 31.6 percent acetone and then filtered at 3° to 6°C. After a few hours, m'ease crystals form in the filtrate and are removed by centrifugation. Recrystallization is satisfactorily accomplished - 13 - from aqueous citrate-acetone as described by Bounce (l9hl). Vhrner in a recent review (1960) summarizes the molecular prOper- ties, the specificity, and the kinetics of urease. The molecular weight of jackbean urease (Sumner gt; 21-: 1938) and bacterial urease (Seneca gt_ g_1_., 1962) was reported to be h73,000. Setlow (1952) reported that the molecular weight per active site, as determined by deuteron inacti- vation, is about 100,000. Hand (1939) concluded from diffusion rate studies that proteins of a weight as low as 17,000 carry urease activity. The study of jackbean urease in monolayers indicated that complete in- activation results from unfolding to form a monolayer (Langmuir and Schaefer, 1939). Setlow (1952) using dry heat inactivation studies con- firmed that the molecule is not unfolded in the active state. The isoelectric point of urease was reported to be 5.0 to 5.1 (Sumner, 1951). Sumner and Poland (1933) demonstrated that twice recrystallized urease gave a nitrOprusside test. From this, they concluded that there were sulfbydryl groups present in the urease molecule. Others have con- firmed this conclusion (Hellerman 33 21,, l9h3; Corin g§.gl,, 1962). Hellerman g§_gl, (l9h3) reported evidence for the existence of three types of mercapto groups of differing reactivity in urease. According to their study, a unit of h73,000 molecular weight contains: 1) 20 to 22 highly reactive groups which are not concerned with enzymatic activity; 2) a like number which are less reactive, but still able to combine with para-cbloromercuribenzoate and.which are essential for enzymatic action; 3) and.a third group, possibly as many as 60 more groups, which react only after denaturation of the protein in concentrated guanidine hydro- chloride solution. Gorin gt_gl. (1962) using para-cbloromercuribenzoate found 50 mercapto groups per molecule of enzyme, which compares fairly - 1h - well with the value 1+0 to m. found by Hellerman gt a}: (1913), Their results, however, do not support the estimate of Bellerman gt 51. (l9h3) regarding the existence of some 60 groups of low reactivity besides the 1&0 to M groups. The relative effectiveness of various metal ions as inhibitors of urease was show: to be: Ag+ > as“ > Cu++ > ed++ > Co++ > m++ > MnH , with 15b++ and re“ unassigned but less than ou++ (Shaw, 195k). This order correlates with the relative insolubility of the sulfides of these metals. The various "activators" or protectors of urease (proteins, amino acids, gum arabic) function by binding heavy metals, thereby pro- tecting the urease sulfhydryl groups (Varner, 1960). There are at present no data which allow any definite conclusions about the molecular weight or the number of active sites per molecule of urease. One group of data suggests a molecular weight of 100,000 per active site, while another group of data suggests that the molecular weight per active site is about 20,000 (Varner, 1960). Sumner (1951) stated: "Urease is absolutely specific. Hundreds of compounds have been tested and have been shown not to be m'drolyzed by urease; among these were various substituted ureas and related compounds." The effects of temperature, pH and substrate concentration on the rate of urease-catalyzed hydrolysis of urea are complex and interrelated (Varner, 1960). wan and Iaidler (1953) showed that the rate of urea m'drolysis increased with increasing substrate concentration, until at very high urea concentrations, the rate actually decreased. This high concentration effect may result from an inhibition of the reaction by amonium ions or from an inhibition by urea. In mnl sulfate buffer the pH activity curve of Jackbean urease showed a sharp optimum at pH 8.0 lTristUdroxymethvlaminomethanc - 15 - (Hall and laidler, 1953). Sumner (1951) had previously reported the pH optimum to vary from 6.1: to 7.6 as a function of buffer ions and substrate concentration. 1". Physiological Role of Urease Numerous workers have speculated about the physiological role of gastric urease. bthews (1920) postulated that urease was involved in hydrochloric acid secretion by the stomach. Others have supported this view, suggesting that amonium ions, derived from urea by the action of urease, lead to the formation of hydrogen ions (Cardin, 1933; than and Minn, 1939; Click , 19149). On the contrary, however, isolated gastric mucosa of whole stomachs from frogs, toads, mice, rats, cats, dogs and skunks can secrete acid in the absence of added urea or ammonia (Davies and Kornberg, 1951; Kornberg and Davies, 1955). It has also been sham that the secretion of hydrochloric acid by mouse stomachs and frog gas- tric mucosa is not increased by the addition of either urea or ammonium chloride to the media (Davenport and Jensen, 1949; Davies and Kornberg, 1951). This and other evidence led Kornberg and Davies (1955) in a re- view of gastric urease research to state: "The evidence thus appears conclusive that gastric urease plays no role in the mechanism of forma- tion of hydrochloric acid by the stomch." It has been frequently suggested that gastric urease plays a role in protecting the mucosa from attack by acid and pepsin (Luck, 1921M; FitzGerald, l9h6; Glick it. 11:, 1950 ; Conway, 1953). Conway (1953) points out that urea therapy of peptic ulcers has been shown to be a valuable therapeutic procedure, and particularly so in the treatment of ulcer cases with dyspepsia resistant to standard medical treatment. Even though the ingestion of large amounts of urea has been shown to be - 15 - beneficial in treating ulcer cases (PitzGerald and Murphy, 1950), it appears that gastric urease is not essential to the welfare of the stomach because normal frogs, rats, cats and pigs have been found with no detectable trace of this enzyme (Kornberg and Davies, 1955). Also, it has been shown that the gastric urease activity in cats is completely abolished by treatment with an antibacterial mixture (Kornberg gt_gl., l95ha) and that the ability of the cats to secrete acid was not changed. Kornberg and Davies (1955) concluded their review by stating that gastric urease is of bacterial origin and plays no essential role in gastric physioiogy. 0n the contrary, Conway (1953) stated that gastric urease in the human subject has a functional significance in neutralizing gastric acidity and protecting the mucosa. There is as yet no firmly established function of urease in plants (Vhrner, 1960). In the cotyledons of citrullus, urease concentration changes during growth, showing an initial rise followed by an abrupt drOp to a low value (Williams and Sharma, 195k). Urease and arginase concentration changes are almost parallel during germination in the soybean (Verner, 1960), suggesting that urease is involved in arginine metabolism. It has been presumed that urease allows bacteria to utilize the urea of animal wastes as a source of ammonia (Seneca at 31,, 1962). nyubimor (1956) reported that living, urea splitting bacteria do not secrete urease into the surrounding medium, and that in.Micrococcus 25235 and Proteus vulgaris most of the urease was bound within the bacterial cells and was liberated only after death and decomposition of the cells. - 17 - G. urease and Ammonia Toxicity Parenteral injections of urease have been shown to be toxic to a number of laboratory animals. Carnot 33 21, (1921) found soybean urease highly toxic to dogs. Tauber and Kleiner (1931) reported that jackbean urease was toxic when injected subcutaneously into mice or intravenously into rabbits. They obtained a ML!) of 90 Sumner units per kilogram of body weight for mice. The blood urea was quickly and completely trans- formed into ammonia and carbon dioxide. They suggested that ammonia was the toxic agent since the symptoms closely resembled those exhibited in ammonia poisoning. A large amount of urease was found in the blood one hour after the injections. Kirk and Sumner, the same year (1931), showed that single injec- tions of recrystallized jackbean urease, either subcutaneously, intra- peritoneally, or intravenously were toxic for the rabbit at a level of 27.5 Sumner units per kilogram of body weight. They also showed that a dose of approximately 25 units given intraperitoneally was toxic for a guinea pig. They confirmed other findings that the poisonous effect of injected urease was due to the ammonia produced. landford (1961) produced death in dogs from intravenous injections of 13.5 modified Sumner units per kilogram of body weight. Plasma am- monia and glutamine were elevated while blood urea was decreased. he pointed out that the use of urease afforded a unique method for studying ammonia metabolism ig_!izg, since, within limits, a self-per- petuating, cyclic release of ammonia is achieved. Recently, Deng and Visek (1963) reported the results of single parenteral injections of jackbean urease in nonimmunized animals. The acute 1.050 in Sumner units (10 micrograms of protein per unit) of - 18 - urease per kilogram of body weight were: mice, intraperitoneal (IP) or subcutaeous (SQ), 50 to 60, intravenous (IV), 25; rats, IP, ’48 to 50. IV, 20; rabbits, IP, 25, IV, 6; and guinea pigs, IP, 35. The serum of moribund animals had toxic levels of ammonia, distorted electrolyte levels and depressed urea concentrations. Ammonia toxicity has also been reported to be the cause of death in ruminant urea poisoning. (Dinning gt_al, 19h8; Gallup $3.21!! 1953; Pierson and Aanes, 1959; Lewis, 1960). Dinning 52.21, (19h8) believed that approximately h milligrams per 100 milliliters was the lethal level of blood ammonia in cattle and sheep while Gallup £3 21 (1953) reported death at 8 milligrams per 100 milliliters. Death may occur from a few minutes to several hours following onset of symptoms. A number of symp- toms of urea toxicity (ammonia) have been reported: incoordination, staggering, dullness, frothy salivation, polyuria, labored breathing, and violent muscular spasms (Dinning gt_al., l9h8; Gallup gt_al., 1953; Pierson and Aanes, 1959). AutOpsy of animals has failed to show any characteristic gross tissue changes (Gallup 53.31,, 1953; Pierson and Aanes, 1959); however, a number of general conditions have been observed: congested and edematous lungs, fatty degeneration of liver and kidney, and hyperemia of the mucous membranes of the digestive tract. The deleterious effects of ammonia have been a matter of interest to many investigators in both plant and animal research. when adminis- tered or absorbed at a slow rate, vast quantities of ammonia may be tolerated by most living organisms because of their highly efficient de- toxifying mechanism in which urea, glutamine and asparagine are formed (warren, 1962). In most biological fluids ammonia exists in two forms, ionized and nonionized; the relative prOportions of each are determined primarily by - 19 - the pH of the fluid. Since toxicity depends on the ammonia which enters the organism, and hence the cell, it is of particular importance that cell membranes are relatively impermeable to the ionized form, whereas the nonionized ammonia passes tissue barriers with ease (Milne gt 9;. , 1958). Several ammonium compounds have been used to produce amnia tox- icity in both ruminants and nonruminants (Greenstein 2.2. 31., 1956; nandrord, 1959; Lewis, 1960, 1961; warren 1962). renewing the adminis- tration of ammonium chloride to sheep, Lewis (1961) reported: decreased pH of the urine and blood; increased anaemia, sodium and potassium in the urine; decreased sodium and chloride and increased potassium in blood plasma; increased urea in the urine; and increased ammonia and urea in the blood. Although the toxicity of amonia to the central nervous system is well known, there is little or no factual information on its specificity of action. It has been postulated that excess amonia any disturb the Krebs cycle by using alpha-ketoglutaric acid, thereby blocking the syn- thesis of subsequent members of the cycle (Bessman _e_t_ 21., 195h; McDermott, 1957). Since the metabolism of the central nervous system is more dependent upon aerobic glycolysis than the rest of the organism (McDermott, 1957), any derangement in the Krebs cycle might seriously alter the glycolytic cycle in the brain. Results of Bandford (1959), however, showed no correlation betwaen the blood level of ammonia and alpha-ketoglutaric acid. These results are in agreement with the clini- cal observations of Dawson g3, g. (1957). but contrary to studies of du Ruisseau _e_1_:_ 2;. (1957) and Sumnerskill at 51. (1957) which showed that blood alpha-ketoglutaric acid concentration was significantly de- creased. - 20 - Although the liver plays a major role in ammonia detoxification in both normal and abnormal states, the arteriovenous cerebral and peripher- al ammonia difference noted in clinical states of ammonia toxicity also implies a continuous removal of ammonia by tissues other than the liver (Lawrence §t_al,, 1958). Duds and Handler (1958) and Krebs gt_gl, (19h9) have shown that glutamine synthesis is the primary mechanism involved in the clearance of ammonia fromianimal tissue. flandford (1961) obtained increased plasr ma glutamine levels in dogs given toxic levels of urease intravenously. Apparently the synthesis of urea via the arginine-ornithine cycle is completely independent of the synthesis of glutamine (Bandford, 1959). It has been postulated that blood ammonia levels may not reflect cerebral intracellular concentration, because of the activity of a system that rapidly binds ammonia by the formation of glutamine from glutamic acid which is synthesized at the expense of alpha-ketoglutaric acid. Studies of the fate of ammonia-N15 given by carotid infusion to cats demonstrated that the major mechanism for ammonia removal in the brain is via glutamine formation, whereas in liver it is urea formation (Earl 53, 21,, 1962). Arginine, other members of the Krebs-Denseleit cycle, and related compounds have been used with varying degrees of success to protect against ammonia intoxication (Krebs gt_ 11., 19h9; Greenstein _e_t_ _a_1_._., 1956; Najarian and Harper, 1956; nandrord, 1959; wright and aorwitt, 1962; lowestein gt 2.1:: 1963). Erecinska and HOrcel (1963) recently reported that glutamate re- versed the inhibitory effect of ammonia on respiration of rat-liver mitochondria by preventing animation of alpha-ketoglutarate. Succinate also abolished the inhibition, but unlike the action of glutamate, - 21 - succinate allowed reductive animation of alpha-ketoglutarate to proceed at a high rate with no depression in oxygen uptake. Although the evidence is not conclusive, it appears that ammonia interferes with aerdbic glycolysis by using alpha-ketoglutaric acid, thereby retarding reactions in the Krebs cycle. thdford (1959) sug- gested that studies of tissue changes in addition to blood changes are in the required direction to resolve the problem. 8. Urease Immunization Kirk and Sumner (1931) using crystalline jackbean urease to study immunologic reactions were probably the first to clearly demonstrate that the serum of rabbits immunized with crystalline urease contained anti- bodies which inhibited the hydrolysis of urea by urease ig_git£g_and ig_ 2132: They gave rabbits gradually increasing doses of jackbean urease intraperitoneally over a period of 60 days. At first, the injections were given every 8 days, but during the last 30 days, injections were given every 2 or 3 days. The initial injection contained 2.2 Sumner units per kilogram of body weight, while the final injection contained about 220 units per kilogram of body weight. Rabbits immunized with urease were found to withstand 100 times the amount of urease found to be fatal to normal rabbits and to show no rise of blood ammonia. urease was injected either intraperitoneally or intravenously. They found that the most rapid immunization in rabbits was obtained by giving intraper- itoneal injections of a urease-antiurease mixture. Serum from immune rabbits was shown to confer passive immunity to normal rabbits and guinea pigs. It was also shown that rabbits severely poisoned by toxic doses of urease could be restored to normal by intravenous injections of antiurease. - 22 - Little work.was done with urease immunization until Dang and Visek (1960), Visek and Dang (l960a,b), and Harbers st 91. (1962, 1963b) re- ported increased growth rate and improved feed efficiency of rats and chicks immunized with jackbean urease. Deng and Visek (1960) injected rats intraperitoneally and chicks subcutaneously thrice weekly with re- crystallized urease for a h-week period. In rats, the starting dose was 10 units per kilogram of body weight, progressing to 25 units per kilo- gram of body weight. The chicks received about the same level of urease. The controls in each case received an equal volume of 0.85 percent NICl. In both rats and chicks there were no differences in body weight during the 0 to h week injection period. But during the h to 8 week period, the immunized chicks gained significantly faster (P‘< 0.01) and were more efficient in converting feed to weight gain. In two successive experi- ments immunized rats grew slightly faster and showed a significant im- provement in feed efficiency. Antibodies to urease were demonstrated in sera of immunized rats by i§_vivo methods with urea-01h andbvisiiise. tests. However, they were unable to detect the presence of antibodies in sera of the immunized chicks. The urea splitting activity of the gastrointestinal contents of immunized animals averaged ho percent less than that of controls. ‘Visek (1962) postulated that the improved performance of rats and chicks immunized to jackbean urease is due to a decrease in ureolytic activity of the gastrointestinal tract. This reduction in urea breakdown, caused by an antiurease-urease reaction, results in a decreased amount of ammonia which the body must detoxify, and thereby results in a saving of energy to the animal. The energy saved then contributes to growth. This hypothesis is based upon the following scheme of urea metabolism. urea - 23 - in the gastrointestinal tract is hydrolyzed into carbon dioxide and am- monia by urease. The lungs exhale the carbon dioxide while ammonia must be detoxificd by other methods which require energy. From the gastroin- testinal tract, the ammonia may go to the liver where it is formed into urea, or it may react with glutamic acid to form glutamine in the periph- eral tissue. The major portion of the urea is removed through the kid- neys, while a portion returns to the gastrointestinal tract. Energy is required in forming urea and glutamine. Two moles of ATP are required per mole of ammonia via the urea cycle and one mole of ATP per mole of ammonia via the glutamine route. III. EXPERIMENTAI.PROCEDURES A. Immunization 1. General The Objective of this experiment was to study the effect of urease immunization upon the performance and hematology of growing pigs. Three trials utilizing,81 growing pigs were conducted in this investigation. Trial I was started June h, 1962 and was terminated September 10, 1962. Trial II was conducted during the period from February 7, 1963 to April 7, 1963. Trial III, the concluding study, was conducted from my 10, 1963 to July 22, 1963. Four levels of urease were used in Trial 1 to determine the level or levels of urease which would be effective. Due to a limited supply of urease, only three pigs were used at the high urease level, whereas the other levels had six pigs each. urease used in Trial I was prepared from jackbean meal by the author. The method of preparation will be described later. Trial II, with ten pigs per treatment and three levels of urease, was conducted to resolve the level of urease that would produce a signif- icant response. A commercial urease powder (Sigma type II) of high urease activity was used in Trial II. A later study of this urease showed that it contained high hemagglutinative activity as well as high urease activ- ity. ‘Effects of urease in Trial II were, therefore, confounded with the effects of the hemagglutinin. A third trial with three levels of urease was conducted to determine the urease level which would be effective. -21.- - 25 - To avoid hemagglutinative effects, urease prepared by the same method as in Trial I was used in Trial III. Yorkshire and Hampshire-YOrkshire crossbred pigs of both sexes were used in these trials. All pigs were randomly allotted with sex and weight balanced. The pigs were maintained in concrete-floored pens and had free access to water and feed at all times. Feed and growth data were col- lected at twoaweek intervals and, in some cases, one-week intervals. Composition of the basal ration which was used in all three trials is shown in Table 1. Blood samples were obtained from the anterior vena cave of all ani- mals by the technique described by Carle and Dewhirst (19h2). Blood (1h milliliters) taken at each sampling period was divided equally for plasma and serum. A combination of sodium fluoride and ethylenediamine- tetraacetate (EDTA) was used as an anticoagulant for plasma samples. Sodium fluoride was used especially to inactivate enzymes. An additional 2 milliliters of blood was placed in a heparinized vial when hematocrit and hemoglobin values were determined. A11 tubes were tightly corked except when they were being sampled. Serum samples were "rimmed" in the tube’and left at room temperature for one to two hours. Separation of both serum and plasma from cells was completed in an International centri- fuge, size 2, model V, at 2000 x g for 20 minutes. Serum was removed, placed in vials and frozen for later determinations of serum antiurease and antihemagglutinative (Trial II only) activity. At the time of slaughter, three-inch intestinal sections plus con- tents were taken from the cranial end of the duodenum and from the caudal end of the ileum. The sections with contents in place were tied off with strings and then removed by cutting on the outside of the strings. With - 26 - TABLE 1 IMMQNIZATION STUDY. COMPOSITION OF RATIONS‘ Ingredients Basal l.25$ urea 2150i urea _3.h0§_urea ‘ Percent Percent Percent Percent Corn 75.30 80.95 87.10 91.60 Soybean meal 20.00 13.00 5.50 - Alfalfa meal 2.50 2.50 2.50 2.50 Dicalcium phosphate 0.80 0.90 1.20 1.30 Limestone 0.60 0.60 0.10 0.h0 Trace mineral saltb 0.50 0.50 0.50 0.50 a vitamin conc ntratec 0.10 0.10 0.10 0.10 312 supplement 0.15 0.15 0.15 0.15 .A and n mixturee 0.05 0.05 0.05 0.05 ”1‘88 - 1025 2050 30180 QAll rations contained 17 percent crude protein, by calculation and analysis, and 0.50 percent calcium and 0.h7 percent phosphorus by calcu- lation. . bContained0.01 percent cobalt, 0.005 percent copper, 0.007 percent iodine, 0.15 percent iron, 1.2 percent manganese, 0.8 percent zinc (high) and 97 percent salt. cSupplied 2 grams riboflavin, h grams d-patothenic acid, 9 grams niacin, and 90 grams choline chloride per pound of concentrate. dContained 6 milligrams of vitamin 812 per pound of supplement. eContained 3,632,000 10 of vitamin A and 800,000 IU of vitamin D per pound of mixture. strings left in place, the sections with contents were frozen until de- terminations of ammonia and ureolytic activity were made. The data were treated statistically by the analysis of variance (Snedecor, 1956). Treatment means were compared by the multiple range test of Duncan (1955). 2. Trial I Twenty-one Yorkshire and crossbred pigs with an average weight of 39 pounds were injected intraperitoneally with the following levels of twice recrystallized ureasel; 0 (saline), 0.1, l and 10 modified Sumner units (SU) per pound of body weight. There were 6 pigs per treatment with the exception of the 10 unit treatment in which only 3 pigs were 1urease was prepared from jackbean meal by the author. - 27 - used. All pigs except those in the 10 unit treatment were injected thrice weekly for h weeks. Pigs in the 10 unit treatment received only six injections. Size of subsequent injections was determined by arith- metic progression. a, a+d, a+2d, a+3d a+(n - l) d where: a is the first injection with values of 0, 0.1, 1 and 10; d is the common increase with values of 0, 0.1, 1 and 10; n is the number of the injection with values from 1 to 6 for 10 unit treatment and l to 12 for other treatments. weekly weights were recorded in the early part of the trial and these weights were used in determining the dose size. Blood samples were taken at 0, 8, 15, 22, 36, 50, 6h, 69, 72, 83 and 97 days for ammonia, urea and antiurease activity determinations. Only 1:1 355 84. 72 and 97-day values will be reported as they are representative of samples gathered on the other dates. 0n the first and fifteenth day of the trial, additional blood samples were taken from three pigs in each treatment at 1 and 6 hours (1 and h hours on the fifteenth day) after the urease injection to study plasma ammonia and urea behavior. On the fif- teenth day, urine collections were taken 2h hours before and after the urease injection. The same three pigs in each treatment were used for urine collections. Pigs were removed thrice daily from the metabolism crates and allowed to eat and drink. urine was collected in large bot- tles containing 10 milliliters of 25 percent sésoh. One-half of the pigs on each treatment were given an additional in- jection of urease after 62 days on experiment (35 days after the twelfth injection) to study anamnestic response. Intestinal sections plus con- tents were taken from the pigs at the time of slaughter. - 28 - 3- his; 11. Thirty crossbred pigs with an average weight of 66 pounds were ran- domly divided by weight and sex into three equal groups. They were in- jected intraperitoneally with the following levels of urease2; 0 (saline), 0.1 and 1 modified 80 per pound of body weight. Urease dose was based upon weekly pig weights and was increased by arithmetic progression as described in Trial 1. The urease injection schedule was modified after five urease injections because of an apparent severe effect of the urease injections on growth and feed consumption. Injections were discontinued after the sixth injection. Investigation of the commercial urease powder showed it to have high hemagglutinative activity. This will be discussed in detail in the RE- SULTS AND DISCUSSION section. At the end of h? days, each treatment group was randomly divided into two equal groups. One-half of the pigs on each treatment received a ration containing 1.25 percent urea (Table 1). The urea ration was made by replacing part of the soybean meal with urea and the total nitrogen was adjusted to that of the basal ration. At the end of 16 days, the urea was increased to 2.5 percent by replacing more of the soybean meal (Table 1), and the total nitrogen level was kept the same as the basal ration. After two weeks on 2.5 percent urea, all soybean meal was re- moved and urea was added to balance total nitrogen. This ration con- tained 3.h percent urea. Blood samples were taken at 1h, 29, h? and 77 days for urea,ammonia,anti- urease activity and antihemagglutinative activity determinations. Hemato- crit and hemoglobin values were determined at 1h and 29 days. 2Sigma type II urease powder was used. - 29 - h- 1.21212}; Thirty Yorkshire and crossbred pigs with an average weight of 31 pounds were injected intraperitoneally with the following levels of twice recrystallized urease3, 0 (saline), 0.5 and 5 modified SU per pound of body weight. There were 10 pigs per treatment. Urease dose was based upon weekly pig weights and was increased by arithmetic progression as described in Trial 1. Injections were made on the following days: 2, h, 6, 8, 11, 1h, 18 and 25. Blood samples were taken at 28, 56 and 73 days for urea, ammonia and antiurease determinations. At the end of 56 days, each treatment was randomly divided into two equal groups. One-half of the pigs on each treatment received a ration containing 3.h percent urea (Table l) for the remainder of the trial. Intestinal sections plus contents were taken at the end of the trial. 5. Urease a. Preparation. Urease was extracted from finely ground defatted h jackbean meal using the method of Sumner (1926), and Kirk and Sumner (l93h). It was recrystallized twice using Dounce's procedure (19hl). The extraction mixture was prepared by mixing 160 milliliters of reagent grade acetone and 3&0 milliliters of cold deionized water containing 10'3 M EDTA. The use of cold water kept the mixture below 28°C. The 32 percent acetone-water mixture was added to 100 grams of jackbean meal in a 1 liter beaker. The mixture was stirred for 5 minutes and then fil- tered through Eaton-Dikeman No. 5&1 filter paper into a 500 milliliter EUrease was prepared from jackbean meal by the author. Jackbean meal used in Trial I was purchased from Sigma Chemical Co., St. louis, Mb. and from General Biochemicals, Chagrin Falls, Ohio for Trial III. -30.. Erlenmeyer flask which was iced after approximately 150 milliliters of filtrate had been collected. The total amount of filtrate was 350 to #00 milliliters. The filtrate was then put into large glass containers which were kept in the cold room at h°c. After at least 2% hours in the cold room, a white sediment was present in the bottom of the containers. The upper part of the liquid was decanted and left for additional crystal- lization to occur. Up to four "crOps" of crystals were obtained from the filtrate over a period of about five weeks. The remaining liquid con- taining the precipitate was centrifuged at 2000 x g in the cold room for 50 minutes (International centrifuge, size 2, Model V). The clear centri- fuge was decanted and the tubes containing residue were allowed to drain in the cold room until the smell of acetone was no longer evident. How- ever, the residue was not allowed to dry out. The residue was dissolved in 6 milliliters of EDTA water and centrifuged at 20,000 x g and h°C for 30 minutes (lourdes Beta-Fuge, Model A). The supernatant contained the urease, and the precipitate contained the impurities. To the crude urease solution was added 0.05 volumes of 0.5 M citrate buffer 5, pH 6.0; and after thorough mixing, ice cold acetone was added slowly with constant stirring to make a 25 percent acetone solution. The mixture was then allowed to stand for at least one week in the cold room. Crystals were separated by centrifuging at 2,000 x g and hOC for 1 hour. The crystals were then dissolved in EDTA water and centrifuged at 20,000 x g and h°c for 30 minutes. The supernatant contained the urease. The same procedure was used for the second recrystallization as was used for the first recrystallization. Crystals from the second 5Citrate buffer 0.5 M, pH 6.0 - Approximately 5 milliliters of 0. 5 M citric acid was added to 95 milliliters of 0-5 M sodium citrate. The pH was adjusted to 6.0 with 0.5 M citric acid. Deionized water was used. The buffer was stored in the cold room in a bottle with a nonmetal cap. - 31 - recrystallization were dissolved for injection in a 0.85 percent NaCl solution. Although this method of urease preparation seems rather simple, the results were often quite variable and disappointing. As pointed out in the literature review, urease is rapidly inactivated by ions of heavy metals and is sensitive to temperature and pH. Deionized distilled water with 10'3M EDTA was used in all steps of preparation. Vigorous mixing was avoided. A11 stages of preparation, except initial extraction, were carried out in the cold room at too. As previously mentioned, additional "crops" of crystals were obtained by allowing the centrifugate to stand in the cold room. The additional "crops” proved worthwhile as they yielded as much activity as did the first "crap". b. £32235 activity. One unit of urease activity has been defined by Sumner and Graham (1925) as that amount of enzyme which will liberate l milligram of ammonia nitrogen from a urea-phosphate solution6 pH 7.0 in 5 minutes at 20°C. This is now known as a Sumner unit (so). A modi- fied SU was used in this experiment so that the assay could be conducted in a water bath (27 i 0.500) at room temperature. All other conditions of the definition were the same. urease activity was determined by measuring the amount of ammonia liberated by mixing Nessler's reagent7 directly with the acidified reaction mixture (Sumner, 1951). A 1.0 milliliter aliquot of the diluted urease solution (usually a 1:1000 dilution of urease solution with phosphate buffer, pH 7.0) to be 6Urea phosphate buffer - Three grams of urea, 6.8 grams of NthPOu and 2.8 grams of xnépoh were dissolved in deionized water and adjusted to 1 milliliters with deionized water. Nessler's reagent - Fifteen milliliters of deionized water and 15 milliliters of Nessler's stock reagent (Polin a'Wu) were mixed. with constant stirring, the mixture was added to 70 milliliters of standard- ized 10 percent NaOH. The reagent was prepared fresh for each assay. - 3g - tested was placed into a 100 milliliter volumetic flask which was im- mersed in a water bath at 27 i 0.500. The 3.0 percent phosphate buffer was also kept in the water bath. At the start of the assay period, 1.0 milliliter of urea phosphate buffer was added with rapid mixing. The reaction was allowed to proceed with mixing for exactly 5 minutes. It was then stOpped by quickly adding 1.0 milliliter of l N HQSOh and mixing. About 80 milliliters of deionized water were added and 5 milliliters of Nessler's reagent were then blown into the flask and mixed. The solu- tion was then diluted to the mark with deionized water. A blank was made in which only the urease was omitted. The Optical density at h80 millimicrons was then determined in a Bausch and Lamb Spectronic 20 and compared to the Optical density readings produced by an ammonia standard. c. Remagglutinative activity. Hemagglutinative activity was based upon the highest dilution of urease which would agglutinate porcine erythrocytes. A 0.2 milliliter quantity of urease solution was serially diluted and tested with an equal volume of a 2 percent suspension of erythrocytes.8 Tubes were mixed and incubated at room temperature over- night. A positive pattern consisted of a thin layer of uniformly agglu- tinated cells covering the bottom of the tube. A negative pattern con- sisted of a red, compact button of red blood cells in the center of the bottom of the tube. The hemagglutination titer was designated as the highest dilution of urease solution in which a positive pattern was Ob- served. 8Heparinized erythrocytes from the control pigs were washed with 10 volumes of 0.85 percent NhCl at room temperature h times and then the cell concentration was adjusted to 2.0 percent with 0.85 percent RhCl. -33- 6. Biological Determinations a. Amnia 1.. Plasma ammonia N, urine amonia N, and ammonia N as a product from urease action on urea, were determined by the microdif- fusion method (Conway, 1957). The Shrink modified Conway units were used (abrink, 1955). Shrink's modification of the Conway microdiffusion unit avoids the use of "greasy" top fixatives by having an extra chamber (closing chamber) which is half-filled with the same solution as used in the outer diffusion chamber (middle chamber) to liberate the ammonia. The absorbing fluid is contained in a center or inner chamber. Plasma or urine ammonia N was determined as follows. One-half mil- liliter of plasma or 0.25 milliliter of a 1:50 dilution of urine was placed in the middle chamber of the unit. The center chamber contained 1.0 milliliter of 0.5 percent boric acid indicatorg. One milliliter of h5 percent potassium carbonatelocontaining 0.025 percent NPX Tergitol was then placed in the middle chamber on the opposite side from the plasma (or urine) so that the two did not mix until the tOp was in place. Then approximately 1.5 milliliters of the same potassium carbonate solu- tion was placed in the closing chamber and the lid was put in place. The plasma (or urine) and potassium carbonate were then mixed thoroughly and the units were incubated at h0°C for 1.0 hour. (Incubation time should be uniform with all samples.) units were removed from the warm room and the tops were removed carefully so as not to drop potassium “ .4 _W —v ’— 9Boric acid indicator 0.5 percent - Five grams of boric acid were dissolved completely in 260 milliliters of ethanol and 700 milliliters of deionized water. Thn milliliters of a mixed methyl red-bromcresol green indicator (Methyl red, 0.066 grams, and bromcresol green, 0.033 grams, were dissolved in 70.0 milliliters of ethanol and then adjusted to 100 milliliters with deionized water) was added to the boric acid solution and brought to the desired red color with weak Naon. Then 0.025 percent NPX Tergitol (Onion Carbide Chemical Co.) was added and the volume was adjusted to 1000 milliliters with deionized water. 10Potassium carbonate was boiled for 15 minutes to free the solution of ammonia. - 3h - carbonate into the center chamber. Contents Of the center chamber (now green) were then titrated with approximately 0.002 N 8250“ from a micro- burette until the first permanent pink color appeared. The concentration of unknowns was calculated from ammonia standards which were analyzed at the same time. All determinations were performed in duplicate and a reagent blank was also used. Determinations of plasma ammonia were made within 2 to 3 hours after the samples were taken. b. Serum antiurease activity. Determination of antibody production was based upon the ability of the immune serum to inhibit the power of urease to hydrolyze urea (Kirk and Sumner, 1931). This is called anti- urease activity. The method is given below. All reactions were carried out in the Shrink modified Conway unit. One milliliter of 1.0 percent boric acid indicator11 was placed in the center chamber and 0.25 milliliter of serum was placed in the middle chamber. One-half milliliter of ureaseephosphate buffer solution (1.0 modified 80 per milliliter) was then placed in the middle chamber. The top was put in place (without potassium carbonate) and the serum and urease were thoroughly mixed and the unit was placed in the warm room at hOOC for ho minutes. After incubation, 0.5 milliliter of 3.0 percent urea-phosphate buffer was added quickly to the serum-urease mixture and thoroughly mixed. This reaction was carried out at room temperature (2h to 25°C). Exactly 5 minutes later, 1.0 milliliter of us percent potassium carbonate was carefully and quickly added to the mixture. The top was then sealed with 1.5 milliliters of potassium carbonate solution and the samples were mixed and placed in the warm room for 1.0 hour. 11One percent boric acid indicator was prepared the same as 0.5 percent boric indicator except 1.0 percent boric acid was used instead of 0.5 percent. - 35 - Ammonia produced was determined by titrating the contents of the center chamber with standardized 3280“, which was about 0.02 N. Controls were carried out on all samples in order to determine the amount of ammonia produced by the action of urease upon the serum urea. Phosphate buffer was added to the controls instead of urea-phosphate buffer. Control values (serum +urease +-buffer) were substracted from urease activity values (serum +-urease +-urea). The difference between the orig- inal urease activity (urease +-urea) and the activity after incubation with serum represented the antiurease activity. Kirk and Sumner (1931) defined one unit of antiurease activity as that amount of antibody which will inhibit one unit of urease activity. All samples were determined in duplicate. A preliminary test showed that a urea blank and a urease blank were not necessary. c. Preparation of intestinal sections. Intestinal sections were prepared for determinations of ammonia and ureolytic activity as follows. Frozen tissues were placed in the cold room (#00) 15 to 30 minutes be- fore needed. Ends of the section including strings were cut off and the remainder of the section was cut into small pieces and placed in a semi: micro container. A minimum amount of deionized water was added and the sample was homogenized in the cold room for 1 minute at high speed on a two-speed Haring Blendor, Model NO. PB-S. The homogenized sample was removed and the container was rinsed with a minimum amount of deionized water. The homogenate including washings was centrifuged at 2000 x g for 50 minutes in the cold room. The supernatant was separated by fil- tering through two layers of cheese cloth. Only the supernatant was saved for assay, as Visek egg 5;. (1959) indicated no difference per volume in ureolytic activity between the entire homogenate or its supernatant. - 36 - A 10 milliliter aliquot of the supernatant was taken for dry matter determination at 100°C for 15 hours. Ureolytic activity and ammonia were determined as rapidly as possible after thawing of the tissues. d. Intestinal ggmonia and urease activi_y. One milliliter of in- te stinal supernatant was placed in the middle chamber of the Shrink modified Conway unit which contained 1.0 milliliter of 0.5 percent boric acid indicator in the center chamber. Then 0.5 milliliter of 3.0 percent urea-phosphate buffer was added and mixed with the intestinal supernatant. The reaction was allowed to proceed in the warm room (hOOC) for to min- utes. One milliliter of h5 percent potassium carbonate was then added carefully and quickly to the mixture. The top was then sealed with 1.5 milliliters of potassium carbonate solution and the sample was mixed with the potassium carbonate and placed in the warm room for 1 hour. Ammonia produced was determined by titrating the contents of the center chamber with standardized 3260“, which was about 0.002 N. Controls using phos- phate buffer instead of urea-phosphate buffer were carried out to deter- mine the ammonia content of the intestinal supernatant and to correct urease activity values. urease activity was expressed as modified 80 per gram Of supernatant dry matter. Ammonia was expressed as ammonia N per gram Of supernatant dry matter. All samples were carried out in duplicate. e. §ggum_antihemagglutinative activity. 1) Serial dilution of serum. Serum antihemagglutinative activity was based upon the highest dilution Of serum which would inhibit hemagglutination. A 0.2 milliliter serum sample was serially diluted and incubated with an equal volume of a 1:500 dilution of Sigma type II urease (5 grams in 20 milliliters of H20) for 1 hour at room temperature (2h to 25°C). Then 0.2 milliliter of a 2.0 percent suspension of erythrocytes was added, mixed and incubated at room - 37 - temperature overnight. The tubes were read in the same manner as for the hemagglutinative activity test. The serum titer of antihemagglutinative activity was the highest dilution of serum in which hemagglutination was completely inhibited. 2) Serial dilution of urease. Serum antihemagglutinative activity was based upon the lowest dilution of urease in which agglutination was inhibited. In this test, a 0.2 milliliter urease sample (Sigma type II, 5 grams in 20 milliliters of 320) was serially diluted and incubated with an equal volume of a 1:5 dilution of serum at room temperature. One hour later, 0.2 milliliter of a 2.0 percent suspension Of erythrocytes was placed in the tubes and mixed. These were incubated at room temperature overnight and read in the same manner as for the hemagglutinative activity test. The serum titer of antihemagglutinative activity in this test is the lowest dilution of the urease in which there is complete inhibition Of hemagglutination. f. Hematocrit, hemogldbin 22§.E£SE.!;. The hematocrit values were determined by the procedure outlined by McGovern gt 51, (1955). Demo- globin was determined by the cyanmethemogldbin method described by Crosby gt 5;. (195k). Brown's para-dimethylaminobenzaldehyde method (1959) was used for urea N. A 1:50 dilution of urine was used for urea N. B. Toxicity 1. General The Objectives of this experiment were to study the effect Of a large intraperitoneal injection of urease on growing pigs and its rela- tionship to ammonia toxicity. Twenty crossbred pigs weighing 20 to 30 pounds were used in three trials. Two pigs, a control and a treatment, were used in Trial I to determine if a single large inJection Of 100 -38- modified SU per pound of body weight was toxic to the pig, and if the blood urea and ammonia Level were changed. In Trial II with two pigs per treatment, three levels of urease, 0 (saline), 50 and 100 modified SU per pound of body weight, were given intraperitoneally in a single dose. Trial III consisted of two treatments, 0 (saline) and 75 modified 30 per pound of body weight, with six pigs per treatment. Twice re- crystallized urease prepared from jackbean meal by the author (Part.A) was used in Trials I and II, while Sigma urease powder, type II, was used in Trial III. All pigs were placed in metal metabolism crates approximately 20 hours before they were injected and were left in the crates after they were injected. The pigs in all trials were fasted during the entire eXperimental period, but were allowed access to water thrice daily. Urine was collected, in large bottles containing 10 milliliters Of 25 percent agson or HCl, before and after the injection of urease. Blood samples were taken before the pigs were placed in the crates, before the urease was injected, and at specific intervals thereafter. Frequency Of blood sampling was greater in Trials I and II than in Trial III. Plasma and serum were Obtained as outlined in Part A. Sterile tubes, corks and vials were used for serum to avoid any possible bacterial contamination which might influence the urease activity. The pigs were weighed at the beginning and at the end Of each trial. Rectal temperatures were recorded at the time of blood sampling. Hemo- glObin, hematocrit, ammonia and urea were determined as described in Part A. ‘A 1:50 dilution Of urine was used for urea and ammonia determi- nations. Liver samples taken from all pigs in Trial II were stored and prepared in the same way as intestinal sections. - 39 - Liver ammonia and urease activity determinations were the same as for intestinal sections except that 0.5 milliliter of liver supernatant was used, and the reaction was allowed to proceed at room temperature for 5 minutes. The same method, using 0.25 milliliter of serum was used in determining urease activity. Pigs which died were autOpsied. The data were tested by the analysis of variance and the t-test (Snedecor, 1956). Treatment means were compared by Duncan's (1955) mul- tiple range test. 2. Biological Determinations a. Glucose. The Nelson-Somogyi microtechnique was used fOr blood glucose (American Association Clinical Chemists, 1953). A Ba(OB)2 super- natant of whole blood was prepared by mixing 0.2 milliliter of whole blood, 1 milliliter of 0.3 n Ba(OB)2 and 1 milliliter or 5 percent ZnSOu. The mixture was centrifuged for 10 minutes at 2,000 x g. One milliliter of alkaline cOpper reagent and 0.5 milliliter Of the supernatant were placed in a Folin sugar tube and mixed. A marble was placed on top Of the tube and it was autoclaved for 5 minutes at 115°C. After cooling for 1 minute in water at room temperature, 1 milliliter of arsenomolybdate reagent was added, mixed and the solution was diluted to the 10 millili- ter mark with water. Optical density was measured at 5h0 millimicrons using a Spectronic 20. Concentration of sugar in the unknowns was calcu- lated from glucose standards carried out simultaneously. All determina- tions were carried out in duplicate and within 1 hour after the blood samples were taken. b. Serum protein. The serum protein was determined according to the method first described by waddell (1956). Five lambda of serum was diluted to 5 milliliters (1:1000) with 0.85 percent Na01. A reading at - ho - wavelengths Of 215 millimicrons and 225 millimicrons was made on a Beckman Model DU spectrOphotOmeter. The absorbence at 225 millimicrons was substracted from that at 215 millimicrons. This difference multi- plied by lh.h gave the protein concentration in the serum expressed in grams per 100 milliliters. c. Elgctrgphoresis. The serum protein fractions were separated on a Spinco, Model R, paper electrophoresis system (Spinco Technical Bulle- tin 6050A) at room temperature. The relative intensities of the separated proteins were determined by scanning with a Spinco Model RB Analytrol equipped with two 500 millimicron filters and a B-5 cam. d. Serum sodium and pgtassium. Sodium and potassium determinations were made with a Beckman DU spectrophotometer equipped with a flame attach- ment using an oxygen-acetylene burner. A 0.1 milliliter serum sample was diluted to 10 milliliters (1:100) with deionized water. Both sodium and potassium determinations were made from the same dilution Of serum. The standard solution contained aodium:potasaium in the ratio Of 1:1. Sodium was read at approximately 770 millimicrons and potassium was read at ap- proximately 590 millimicrons. Standard curves were run with every set of samples and were determined in the range of 10 to ho milligrams of potas- sium per 100 milliliters and 100 to 500 milligrams of sodium per 100 milliliters. e. Tbtal nitrogen. A modification of the semi-micro Kjeldahl pro- cedure outlined in the Official Methods of the Association of Official Agricultural Chemists (1960) was used to determine total nitrogen in urine samples. American Instrument Company's semi-micro Kjeldahl equip- ment was used. To a 0.25 milliliter urine Blmple in the digestion flask was added 1.9 grams of K280“, 2 milliliters Of concentrated sésoh and - 1.1 - 1 milliliter of 10 percent CuSOh. A small glass head was added, and the sample was digested for at least 2 hours after the solution had cleared. men samples were cooled slightly (about 5 to 10 minutes) and lo millili- ters of deionized water were added carefully. The solution was mixed and cooled to room temperature. The flask was connected to the distillation apparatus and 10 milliliters of to percent NaOH was added after the dis- tillate receiver, which contained 10 milliliters Of 2 percent boric acid and 2 drOps of 0.1 percent bromcresol green indicator, was in place. Steam was passed through the sample for 7 minutes with the tip of the condenser submersed in the boric acid indicator and 1 minute with the tip of the condenser above the surface of the indicator. The distillate was titrated with standardized 0.05 N HCl.from the blue color back to the original yellow-green color. ,A blank determination was run. r. 3553-; calcium. Methods of Mori (1959) and Appleton _e_t_. 22:. (1959) were combined and modified.by C. L. Zutaut (unpublished data) for the de- termination of serum calcium. Into a 10 milliliter beaker containing approximately 7 milliliters of 0.5 M K03, 0.25 milliliter of serum was introduced. Two drops Of 0.1 percent thymolpthalein in ethanol and a small amount of calcein12 indicator from the tip of a micro spatula were added to the beaker. (The amount Of indicator to be used can be deter- mined by the individual analyst.) The solution was swirled.to dissolve the indicators. ‘The solution of diluted serum, which had a green fluores- cence, was titrated with cum13 in a Sargent SpectrO-Electro Titrator, Model SE using manual control until the green color changed to a light 12Calcein - fluoresceinbismethyleneiminodiacetic acid 13cm solution - Approximately 39 grams of 1,2-diaminocyclohexane-N, N,N',n'-tetraacetic acid (cm) were dissolved in 1 liter of deionized water. This was the stock CDTA solution.and was diluted 100 times for the working solution. -hg- purple at the end point. A standard calcium solution containing 10 milli- grams Of calcium per 100 milliliters and a blank were treated the same way as the specimen. g. Serum glutamic-oxalagetic and glutamicepyruvic transaminase. Determination of serum glutamic-oxalacetic transaminase (SGO-T) and glutamic-pyruvic transaminase (SOP-T) activity was made according to the procedure described in Sigma Technical Bulletin No. hlo (12-61). The Sigma hlo-OP combination kit was used as a source of reagents. IV. RESULTS AND DISCUSSION A. Immunization 1. Trial 1 A summary of feed consumption, average daily gain and feed efficiency data obtained in this trial is presented in Table 2. Pigs in each treat- ment were group fed; therefore, a statistical analysis could not be made on feed consumption and feed efficiency data. In general, feed consump- tion was greatest for the 0.1 and 10 unit treatment groups. The 10 unit treatment group appeared to be more efficient than the other groups at all periods except 79-98 days. Average daily gain was significantly (P -.0.05) greater in the 10 unit treatment group than in the control or the 1 unit group at 0-58, 0-79, 0-98 and 30-58 days. NOte that gain was also greatest in the 10 unit treatment group, though not significant, at 0-30 days. Individual average daily gains, sex, age, initial weights and final weights are given in Appendix Table l. The mean values for plasma urea N and ammonia N and serum antiurease activity are shown in Table 3. Plasma urea N and ammonia N levels between treatments were not statistically different. urea N values tended to in- crease in all groups with age and/or weight; whereas, ammonia N values appeared to decrease. Individual values for urea N and ammonia N are given in Appendix Tables 2 and 3, respectively. Serum antiurease activity of all treatment groups was significantly (P ‘.0.01) greater than the control at 6h and 72 days (Table 3). Anti- urease activity was present at 15 days, though not statistically - h3 - - uh - TRBLE 2 TRIAL I. FEED CONSUMPTION, AVERAGE DAILY GAIN AND FEED EFFICIENCY OF UREASE IMMUNIZED _A;N1_3_ NONIMMUNIZED caowmc PIGS‘b t t Time, days 0:30 30:58 358-79 79- 8 0.58 0-79 0-98 Av. daily feed intake, 1b. Control 2.65 3.68 h.29 5.12 3.22 3.50 3.82 0.1 Unit 3.00 3.87 h.h6 5.51 3.55 3.70 h.10 1 Unit 2.97 3.7h h.29 5.u2 3.h0 3.6a 3.9a 10 Unit 2.83 b.23 b.3h 6.h6 3.67 3.78 h.30 Av. daily gain, lb. Control 1.18 1.56 1.65 1.h0 1.36 l.h6 l.h3 (0.10)°(0.03) (0.08) (0.06) (0.06)(0.06)(0.05) 0.1 Unit 1.3h 1.55 1.69 l.u8 l.hh 1.51 1.50 (0.06) (0.06) (0.05) (0.09 (0.05)(0.0h)(0.0h) 1 Unit 1.27 l.u8 1.59 1.36 1.37 l.h3 l.h2 (0.07) (0.08) (0.10) (0.15) (0.05 (0.0u3(0.063 10 Unit 1.h2 1.77e 1.82 1.60 1.5 1.66 1.6h (0.0u) (0.08) (0.09) (0.13) (0.05)(0.05)(0.06) Feed per lb. gain, lb.f Control 2.26 2.33 2.59 3.63 2.36 2.u3 2.66 0.1 Unit 2.2a 2.u0 2.63 3.72 2.h2 2.h8 2.72 1 Unit 2.33 2.h0 2.69 3.95 2.h8 2.5a 2.77 10 unit 2.00 2.1a 2.37 h.00 2.25 2.29 2.61 IIJ’ackbean urease prepared by the author. bSix pigs per treatment except 10 unit treatment which had only three pigs. .Average initial weight, 39 pounds. §Stsndard error of the mean in parentheses under mean. Significantly larger than the least two treatment means (P 0.05). eSignificantly " " " other treatment means (P “10.05). fPigs group fed. significant because of the large within-group variation, especially in the 0.1 unit group. At 36 days, serum antiurease activity of the 0.1 unit group and 1 unit group was significantly larger than the control group. Although the serum antiurease activity of the 10 unit group was larger than the control group, the difference was not significant at 36 days. As evidenced in Table 3, the maximum urease activity recorded for all groups occurred at 72 days. Note in Appendix Table k that this was true for the pigs which received the additional urease injection at 6h days, as well as for those which received only saline at that time. - hS - TABLE 3 TRIAL I. PLASMA UREA N'AND.AMMONIA N LEVELS, AND SERUM.ANTIUREASE ACTTIVITY 0F UREASE IMMUNIZED AND NONINMUNIZED GROWING PIGS ‘b Time,gdays 0 15, #36 6h 72c .21? Urea N, mg./100 ml. d Control 13.hi1.0 10.6i0.9 l7.hi0.7 27.3:1.3 2h.3 1.2 18.0+0.9 0.1 Unit 1h.7-0.9 12.0+1.3 18.5 1.2 2h.0 1.2 22.2 1.9 17.1.0.7 1 Unit 12.3 0.8 10.2 0.8 18.5 1.1 26.7 1.3 22.8 1.0 17.1 1.7 10 Unit 9.6 1.6 10.8+1.7 15.7 1.9 23.h*3.0 26.8 2.6 18.2 2.2 Ammonia N, mcg./lOO ml. Control 638-u6 856181 780 hl 60h+32 h67~30 577 1h 0.1 Unit 69h-91 758iu6 899 65 5h1+l9 too-35 h9l 19 1 Unit 690 87 836 A3 921 51 557L19 h3h-11 515 32 10 Unit 750*2h 683*110 100k 110 589v2l h73-h5 533.15 Antiurease activity, unit/ml. x 103 Control 0'18 ~111h 3<11 0’9 -7 l3 -h29 0.1 Unit 1:13 uh+32 70.1.e 15826ee 195.19ee 25 25 1 Unit 0 15 61+18 9725ee 173 1Bee 2&6 ahee 62+20 10 Unit -5*h hh*10 53 20 131+8ee 21.1.50ee 57+57 aJ'ackbean urease prepared by the author. bSix pigs per treatment except 10 unit treatment which had only three pigs. Average initial weight, 39 pounds. cOne-half of the pigs on each treatment given an additional in- jection after 6h days on experiment. dStandard error of the mean. eSignificantly greater than the least treatment mean (P 0.05); °e(P‘\ 0.01). Although all pigs had an increase in antiurease activity following either urease or saline injection at 6h days, there appeared to be a greater increase of activity in pigs on the l and 10 unit treatment groups which received urease. Pigs on the 10 unit treatment had the greatest response to the additional urease injection. Serum antiurease activity in treated pigs at 97 days was about the same as at 15 days, and was not signifi- cantly different from the control pigs. It is evident from this data that all levels of urease injected in this trial were effective in stimulating antibody production with some evidence of the 1 unit level being more effective than the 0.1 or 10 unit levels. -l+6- As pointed out in the EXPERIMENTAL PROCEDURES, the measurement of antibody production in these trials was based upon the inhibition of urease by serum. Marucci and Mayer (195h) in quantitative studies on the inhibition of crystalline urease by rabbit antiurease reported that the inhibition of the enzymatic activity of urease by a specific anti- body is only partial. They Observed that mixtures of urease and anti- urease in the equivalence zone displayed about 70 percent of the activity of the enzyme alone, or 30 percent inhibition. Mixtures in the extreme antibody excess region showed approximately 80 percent inhibition. Their data did not support Kirk and Sumners' (1931) idea that decrease of the enzymatic activity of urease when flocculated by antibodies was due simply to the aggregation. Marucci and Mayer (195k) explained the inhibition of urease activity in terms of steric hindrance on a molecular level - the more antibody molecules combined with a molecule of urease, the greater the steric interference. Plasma urea N and ammonia N values following the first and sixth urease injection (15th day) are presented in Table h and Appendix Table 5. The urea N level of pigs in the 10 unit treatment group was signif- icantly (P < 0.01) less than other groups at 1 and 6 hours after the first injection of urease, and the ammonia N level was significantly (P*< 0.01) greater at the same time. Urea N and ammonia N values be- tween groups following the sixth urease injection were not different. Although pigs receiving the 10 unit level of urease did not show symptoms of urease toxicity following the first urease injection, the urea N and ammonia N data do suggest that slightly more ammonia was being produced than could be adequately metabolized by the body. At 15 days, antibody production was probably great enough to tie up most of the urease in- jected, so that plasma levels pf ammonia were not increased. - h7 - TABLE h TRIA1.I. EFFECT 0P INITIAL.AND SECONDARY INJECTIONS 0P UREASE UPON _PLASMA UREA N AND AmoNIA N LEVELS 0P GROWING PIGSab ’; ‘—-—— ————v . lst Dgy_pf Trialc 15th any of Trialc Time, hours 0 1 6— 0 1 3+ Urea N, mg./100 m1. Control 111.016d lu.1i0. 6 13.1i0.6 10.2+1.6 ll.2~2.l 11.0+2.3 0.1 Unit 13.1e0.1 13.510. 5 12.uio.u 11.2:2.h 10.713.3 10.1i3.0 1 Unit 13.3i1.3 13.hil. 0 12.610.5 10.ht0.6 10.3i0.7 lO.6il.S 10 Unit 9.6%1.6 10.5+1. 0f 9.u+0.5f 10.8+l.7 ll.8:1.5 ll.5il.6 Ammonia N, mcg./100 m1. Control 681t63 525:12 553157 8771102 729198 510i33 0.1 Unit 88113.76e 665+8l 501(5h 663:27 902+88 h13i15 1 Unit 591i21 590+L7f 539 26 816i89 735:77 67lilh0 10 Unit 750i2u 96o+56 851.162f 6831111 716i38 57u125 aJ'ackbean urease prepared by the author. bThree pigs per treatment. cBlood samples takza and urease injectiCn made at zero hour, then blold dsamples taken at l and 6 (or k) hours. dStandard error of the mean. eSignificantly greater than the least two treatment means (P + 0.05). fSignificantly different from the other treatment means (P 20.01). Urinary data collected in this trial 2h hours before and after the sixth urease injection (15th day) are shown in Table 5. Urine flow was significantly different between treatment groups following the urease injection. Urine urea N levels of the 1 and 10 unit groups were signif- icantly (P “30.05) larger than urine urea N levels Of the other two groups during the post injection period. Urine ammonia N levels of the 0.1 unit group were increased and were slightly greater than for the control group after the injection. Most Of the ammonia N and urea N difference between the groups is accounted for mainly by the difference in urine flow, as the concentration of urea N and ammonia N per unit of urine was not greatly different between the groups. It would appear from this data that the urease injected pigs had a greater urea N and ammonia N excre- tion rate than the control pigs, which would suggest that urea other than - h8 - TABLE 5 TRIAL I. ERFECT 0F.A SECONDARY INJECTION or UREASE 0N URINE PLow, UREA N, AND.AMMONIA N or UREASE SENSITIZED GRowINC PIQ§_§E§ Treatment Control 0.1 Unit 1 Unit 10 Unit Size of urease injection SU/pound EU 0 0.6 6 60 Urine flow, m1./hr. Pre-injection 3 1 as“ 29.6113 3 Post-injection l 1 2 23a1i2038e Urine urea N, mg./hr. Pre-injection 293m5h 367:37 293x18 368+30 Post-injection 188il7 309:18 352:50e 392179e Change ~1051h2 ~58th5 59:31e 23i5l urine Ammonia N, mg./hr. Pre-injection 16.712.5 16.112.8 17.0:3.2 16.5%1.5 Post-injection 12.9t2.2 23.5th.1 18.0i1.h 17.210.h Change -h.2i2.u 7.ui5.5° 1.0:1.6 0.7i1.l !_—- 4.‘ aJ'ackbean urease prepared by the author. bThree pigs per treatment. cAll pigs placed in metabolism cages 2h hours before and after the sixth urease (15th day of trial) injection. Standard error of the mean. eSignificantly greater than least treatment mean (P < se.a mw.n om.~ me.~ ms.a H:.H mm.a sea as we a m-mma on.a He.a oe.H mm.a ss.m em.~ om.~ can an om a m-oa mm.a >m.a sm.a mm.n mm.” om.a ms.a and mm om a m-oa mm.a ae.n ms.a we.” es.a sm.a sm.a mes mm a m-oo~ mm.a om.~ ss.a ow.a me.~ mm.s sn.a sow mm mm x m-sma mm.a mm.a mm.a om.a ms.a os.a mn.H can me p» z HH-~ can: H.o mod mod mod mod mod mod 3.0 mm Aos.nv Amo.se “om.nv Ame.ne lee.sc mom.ne Amn.nv loose meme ..4 se.a om.n mm.s mm.a mm.H mm.a ms.a «on mm om m m-mma H~.H mm.~ om.a em.” sm.a mo.a osuo men as am a A-mm mm.a mm.a we.“ mm.a mm.a Hm.a an.a men am am a m-mm mm.H mm.a em.a os.~ sm.a mm.a e~.a med mm mm a om-ama mm.a om.a em.a om.m em.a m:.a mm.a own as om : s-oa ee.a w>.a em.a cm.H Hm.a He.a >~.~ Han es cm : m-oH HOhvnOO 2.6 mfio ofio Add; 5.1.»: and son 6. 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AQHUZHBZOOV H mamas anzmmm< TRIAL I. PLASMA - 9h - APPENTIX TABLE 2 UREA N LEVELS 1n IMMUNIZATION swung (n5./100 212° Pig Thme,_days ,‘_ No. 0 15 436 '65 72 3:31 (1:111:21 10-3 18.5 13.0 19.0 29.2 23.6 19.2- lo-h 11.0 7.3 16.6 29.h 2u.u 16.6 151-10 16.6 10.2 15.1 30.7 29.6 21.7 39-3 lu.0 11.8 17.3 22.0 22.3 18.u 39-5 lu.h 12.u 16.2 27.5 2u.3 16.1 155-9 9.8 8.7 19.9 25.0 21.6 15.7 Av. (l3.h) (10.6) (17.h) (27.3) (2h.3) (18.0) St 1.0 0.9 0.7 1.3 1.2 0.9 0.1 Unit 2-11b 13.0 15.8 1h.h 22.5 15.5 18.6 151-8 13.h 10.0 21.6 25.0 2u.h 18.6 200-3 12.9 7.9 19.7 20.1 22.0 lu.9 10-5b 1h.2 11.8 20.1 28.6 27.5 17.6 10-6 15.6 10.6 15.7 20.0 18.2 17.7 151-9b 18.9 15.6 19.7 27.8 25.5 15.h Av. (1h.7) (12.0) (18.5) (2h.0) (22.2) (17.1) SE 0.9 1.3 1.2 1.2 1.9 0.7 1 Unit 3-9b 12.0 lo.h 19.5 29.5 26.0 21.8 3-10 11.9 9.5 20.7 27.0 25.7 22.6 10-2b 15.9 11.1 18.6 22.0 21.5 11.0 39-1 12.5 13.h 20.1 25.h 19.3 16.7 155-7b 10.8 8.9 18.3 27.6 22.7 11.9 155-8 10.8 7.6 13.5 21.8 20.9 15.6 Av. (12.3) (10.2) (18.5) (26.7) (22.8) (17.1) SE 0.8 0.8 1.1 1.3 1.0 1.7 10 unit 39-8b 12.8 1h.1 18.3 29.1 30.0 22.2 150-6b 8.1 9.9 16.8 18.9 22.1 1h.7 150-10 7.8 8.5 12.1 22.2 28.5 17.7 Av. (9.6) (10.8) (15.7) (23.1) (26.8) (18.2) SE 1.6 1.7 1.9 3.0 2.6 2.2 aJ’ackbean urease prepared by the author. bOne-half of the pigs on each treatment were given an additional injection of urease after 6h days on experiment. -95- APPENDIX TABLE 3 TRIAL I. PLASMA AMMONIA N LEVELS IN IMHQEIZATION STQQY (mcg1/100 mllé Pig Time, days No. 0 15 __ jg; 6H 72 __9I__ Control 10-3 556 970 715 512 “02 535 lo-h 760 672 700 571 365 596 151-10 728 988 833 538 529 5&5 39-3 506 567 905 67h 561 591 39-5 73h 860 66h 620 h76 631 155-9 516 1078 865 706 I66 566 Av. (638) (856) (780) (60“) (h67) (577) no 81 1.1 32 30 1h 921.9911. 2-1lb 996 612 829 550 386 520 151-8 918 672 777 5&5 598 sho 200-3 738 706 782 592 h81 515 10-5 1.91. 816 1171 566 1.92 1.09 10-6b ash 83h 1017 538 360 . ugh 151-9b 536 910 818 A52 huh A70 Av. (69%) (758) (899) (581) (#60) (#91) SE 91 #6 65 19 35 19 1 Unit 3-9” 630 931 767 Shh huh 556 3-10 556 682 1110 538 #13 520 10-2b 586 78h 1013 566 A39 Ash 39-h 506 886 8AA 6A6 A28 631 155-7b 772 798 869 522 .75 non 155-8 091 88% 920 528 h02 525 Av. (690) (836) (921) (SS7) (“3%) (515) SB 87 h3 51 19 11 32 10 Unit 3983 708 568 787 630 386 530 150-6 750 got 107% 576 5&0 510 150-10 792 576 1150 561 A92 560 Av. (750) (683) (1008) (589) (#73) (533) as at 110 110 21 us 15 aJ'ackbean urease prepared by the author. bOne-half of the pigs on each treatment were given an additional injection of urease after 6h days on experiment. -96.. APPENDIX TABLE 8 TRIAL I. SERUM ANTI a E ACTIVITY IN UMMUNIZATION STUDY units m1.x _v 3 Pig __1 Time da 8 -: no. 0 15'w 36 6E 72 .91 Control 10-3 6 3h 10 11 38 -20 lo-u -66 -8 -1h 8 -50 -26 151-10 22 -8 -31 -6 -16 -20 39-3 28 -31 h8 -hh o 0 39-5 A6 -10 8 22 16 3h 155-9 -ho 15 -2 7 -30 10 Av. (0) {-1) (3) (0) (~7) (~h) SE 18 1h 11 9 13 9 0.1 Unit 2.11b -8 -1 7h 230 186 68 151-8 -1h 67 62 208 218 66 200-3 -h2 159 91 17h 262 82 10-5 1h 115 70 136 216 -u 10-6b 50 -h0 82 113 139 -76 151-9b 8 -1h ho 89 150 cc 15 Av. (1) (uh) (7o)c (158)c° (195) (25) SE 13 32 u 26 19 25 1 Unit 3-9b -2 35 1A6 196 261 62 3-10 20 70 28 138 253 h2 10-2b -72 133 190 2A2 352 15k 39-11b 1h 29 56 15k 216 10 155-7 27 16 89 122 185 3h 155-8 1h 81 7h 183 209 cc 70 Av. (0) (61) (97)cc (173)°° (256) (62) SE 15 18 25 18 2h 20 10 unit 39-8b -9 2h 65 137 29h 120 150-6b 1 50 80 lul 295 86 150-10 3 57 13 115 1&5 35 Av. (-5) (ha) (53) (131)°° (abh)°° (57) SE n 10 20 8 50 57 'Jackbean urease prepared by the author. bOne-half of the pigs on each treatment were given an additional injection of urease after 6h days on experiment. All other pigs inJected with saline. cSignificantly greater than the least treatment mean (P- 0.05), °¢(P.;0.01). -97.. .hmo.ow.mv memos assausonp or» pasta one cusp nopsoaw havcsofiufinmfimo .mnson A: nov 0 one A as comma one: madmasm moods use convenes ms: masons can» .nson onou as some» one: moaaasm woodmn .aonpss one no coneaona masons :sopxomus 0H 00 em :0 H0 0a 0.0 0.0 s.m 4.0 0.0 H.0 m0 Amasv A0000 A0000 Aflomv Am00v 0A400V AH.0HV As.0nv AN.HHV Ae.mAv A0.mav AH.MHV .>< mm: 000 00s 00: 000 00s «.0 m.» 0.» s.mH 0.0a 0.0a 0-000 :00 00» 050 see 0H0 0H0 H.0 0.0a 0.0a s.ma «.ma :.m~ 0-HOH ems :00. 0.0 0H0 :00 000 0.0a «.02 0.0a 0.0a 0.4a 0.0. Ha-m one: H.0 mm 00 00H 50 «a 00 0.0 ”.0 0.H 0.0 0.0 0.H mm Aoamv A0me. Aesmv A0000 A0000 Aa00v A0.Hav AN.HH0 Am.0av Aa.mav Aa.eav 10.2HV .>< N00 «00 000 0:: 000 00s «.0 5.0 0.0a 0.0a 0.m~ 0.0a 0H-Hmo 0:: 000 000 000 :00 00s m.0 0.0 m.e 0.0a m.sH 0.Hs s-0H mam sa0 0s0 000 0:0 000 0.0a 0.0H 0.0a m.ea 0.0a 0.0H m-0a aoaasoo sh ; has HWIQB 0 03b 05“ 03b .03 an ah .H cab agiim ah H as O 002 7.- I- 00 no a.» 7», (lane sea son some use and 000 .As 00H\nmoe..z onsommd .As 000w.0m .2 not: 1 ommemmmimo mZOHEZH Mgofimm 024 AQHBHZH UZH§OM mch 93:06 ho g x $332 92 I g 54min .H damn. m mumdfi anzmmm< - 98 - .Aao.onmv memos possessa» guano on» song vconouuue maussofiuacwwme .Amo.oV.mv names escapees» on» pmsoa on» can» nopaonw zapaaoauacmamo .mnson A: nov m was A as some» one: madmasm cooHp was anaconda mos mesons con» .aso: one» as some» one: mmamasm 00o~mn .nonpsm one ha monanonn mucous nsooxosua 0m 00 ass 00 00 em 0.H 0.H e.a 0.0 0.H 0.H an :5 05 30000300008000 803 3.3 10.5 10.030300 1.10.0.3 3.00 .5 N00 00» 000 :00 0HOH 000 0.0a 0.HH 0.0 0.0a 0.0 0.» 0H-00H 0N0 ~00 :00 :00 0H0H 00» 0.0 0.0 0.0 0.0 0.0a H.0 0-00m «H0 000 000 0:0 0:0 000 0.0a 0.:H ~.:H . m.ma 0.m~ 0.00 can: on 00H 0» 00 0m 0: Am 0.H 0.0 0.0 0.0 0.H m.~ mm 3.0V 1005 0.00 1003 A0000 :03 10.0: 2.0: 3.0: 0:640 3.3 3.0: .2 0e0 000 :0» :00 000 000 0.0a 0.0m s.aa A.ma «.0m 0.0m 0-0a 00: 000 000 :00 000 000 e.» 0.0 0.0 A.ma «.0H 0.HH 0H-m 0:0 000 400 00: N00 000 0.HH 0.HH 0.0H e.aa 0.HH 0.0a 0-0 can: a .00 an ummsw .ns 0 .00 .ns A .00 0 .as .00 a .ns 0 .nn.0 .00 a .nn 0 .oz and 0004 110.0 ”mm nu0a Assn one 0am .As 00. . on .2 annoaa< .As 00H\.um .z one: ucmmmn z < 00.2 00.2 00.0 00.0 00.0 000 00 00 2 0-002 00.0 00.0 00.0 00.0 20.0 000 :0 20 2 0-002 00.2 00.0 00.0 02.0 00.2 002 00 00 2 0-00 00.2 00.2 00.0 00.0 00.2 000 00 20 2 0-00 00.2 00.0 02.0 02.0 00.0 000 00 00 0 0-00 00.2 00.0 00.0 22.0 00.0 :00 00 00 z 0- 00.0 00.0 20.0 00.0 00.2 000 00 00 a 0-00 00.0 00.0 00.0 00.0 00.0 000 00 20 a 2-00 00.0 00.2 00.0 00.0 00.2 000 00 e0 2 0-00 00.0 00.0 00.0 20.0 00.0 000 00 00 a 2-00 02:: 0.0 00.0 00.0 00.0 00.0 20.0 00 00.20 200.20 200.20 200.00 200.20 22000 2000 .>< 00.2 00.0 00.0 00.0 00.0 202 00 00 2 0-202 00.0 00.0 00.0 00.2 00.0 000 00 H0 0 0.002 00.2 00.2 00.0 00.0 00.2 000 00 20 2 0-00 00.2 00.0 00.0 20.2 00.2 000 20 00 2 0-00 00.0 00.2 00.0 00.2 00.2 000 00 00 2 0-00 20.0 00.0 00.0 02.0 00.0 000 00 00 0 0-00 00.2 00.0 00.0 02.2 02.2 000 00 :0 2 0-00 00.0 02.0 00.2 00.2 02.2 000 00 e0 0 0-20 00.2 02.2 00.2 00.0 00.0 002 00 :0 2 0-00 00.2 00.2 00.0 00.0 00.0 :00 00 00 2 0-00 Hoavcoo 210000-11 00-0 02-00 0000 .02 ..»2 .001..02 0000 200 .02 00022 0000200 00< 020 n 0020000 00000 20H900H20220 20 0000 0202000 00 2020 000<0 m0< 02< .020003_0<200 .000002 0200020 .200 .000 .00 02002 0 00002 20020000 7.— 0|. 400.0 V 5 000008 0.0093600» 03 .0230 :93 000.0 00003000000050 .0000800000 000 a 00009 new .000000 000» 000000 «on: 00000 :00: 0000000 000 «no: 00000 0000 00 0000 no 0000-0000 .uoosoa 00 0000 000000 030000 00.0 00.0 00.0 00.0 00.0 00 00.00 000.00 000.00 000.00 0000.00 00000 0000 .>4 00.0 00.0 00.0 00. 00.0 000 00 00 0 0-000 00.0 00.0 00.0 00.0 00.0- 000 00 00 2 0-000 00.0 00.0 00.0 00.0 00.0 000 00 00 2 0-00 00.0 00.0 00.0 00.0 00.0 000 00 00 0 0-00 00.0 00.0 00.0 00.0 00.0 000 00 00 2 00-00 00.0 00.0 00.0 00.0 00.0 000 00 00 2 0-00 00.0 00.0 00.0 00.0 00.0 000 00 00 0 0-00 00.0 00.0 00.0 00.0 00.0 000 00 00 2 0-00 00.0 00.0 00.0 00.0 00.0 000 00 :0 0 0-00 00.0 00.0 00.0 00.0 00.0 000 00 00 0 0-00 0000 0 0000-. uu00-0 .00-0002 .00 000: 0200 x00 .02 :I- 00:00 0000000 004 000 000020000 00000 200040020220 20 0000 0202000 00 2040 00040 0040004 024 .000002.04200 .000002.0400020 .200 .004 .00 04000 " ‘llll 1‘! 1 ‘ 1 00002002000 0 00040 00020004 APPENDIX TABLE 9 TRIAL II. PLASMA QEEA N LEVE;§.IN IMVUNIZATION STUDY (2%.4100 m1.2a Pig _» Ting, dQXS _No. 1h 29 1_1_ ET 775-— Control 93-7b 17.h 1h.7 23.u 2u.7 9h-7 10.7 11.0 1h.3 16.A 9L9b 16.2 1h.8 16.9 20.9 95-3b 13.2 15.2 16.8 17.1 96-3 17.6 10.9 12.9 13.7 96-6b 1h.3 12.9 16.5 21.9 96-8 15.A 11.9 1u.5 13.9 97-6 19.1 1h.1 20.6 20.2 100-1_ 11.u 13.6 15.5 1u.9 101-6D 17.u 15.8 19.5 2u.5 Av. (15.3) (13.8) (17.1) (18.8) SE 0.9 0.7 1.0 1.0 0.1 Unit 93-1 1h.1 12.7 18.8 15.8 gu-ub 18.5 19.8 17.3 16.1 95-h 12.8 15.2 19.7 17.7 96-1 1h.5 12.3 15.9 16.0 96-9b 13.9 12.7 13.6 20.2 97-2 10.6 13.1 1u.6 20.2 98-3b 15.6 15.5 19.6 21.9 99-3 11.8 1u.8 19.2 20.8 loo-Mb 12.1 17.h 19.7 22.3 100-7 15.8 15.9 16.A 11.1 Av. (1u.0) (1h.9) (17.5) (18.2) SE 0.7 0.8 0.7 1.5 1 Unit 93-3 12.6 18.2 17.9 23.8 95-1 11.3 8.u 1u.0 22.5 95-7b 17.3 19.7 23.5 26.h 96-2 12.9 13.6 16.h 12.h 96-7 17.3 15.1 16.8 19.2 96-11 11.2 11.1 12.3 18.5 97-3b 13.u 15.h 20.0 13.8 98-5 16.0 20.7 17.9 23.2 100-5b 2u.7 15.8 2u.u 31.5 101-1b 11.2 13.1 16.9 18.8 Av. (15.1) (15.1) (18.0) (21.0) SE 1.3 1.2 1.2 1.3 aSigma urease type II powder. bOne-half of pigs in each treatment were fed rations containing urea. See Table 9 for treatments. - 1.7314 .- AFPENDIX TABLE 10 TRIAL II. PLASMA AMMONIA N LEVELS IN IMMUNIZATION sngY (meg;/100 m1.)§ Pig _M*__~_ “_gime, days 11.11 321...... 1...“... .18 Q - 39 ____ '15? 1177? Con+rol 93-7b 700 857 357 815 98-7 658 713 393 862 98-9b 557 531 357 368 95- -3b 687 857 399 391 96-3 879 628 327 509 96-6b 858 868 298 388 96- 8 567 851 637 801 97- -6 510 880 387 377 100- -1b 562 678 321 862 1016 695 558 308 862 Av- (583) (581) (378) (819) SE 28 31 31 8 0.1 Unit 93-1 887 526 363 871 98-8 605 686 578 519 95-8b 516 886 811 888 96-1b 837 685 327 391 96-9b 613 708 887 886 97- -2 890 776 339 862 93.3b 869 668 357 876 99-3 516 776 285 838 100-8b 515 668 363 867 101-7 572 583 883 580 Av. (518) (683) (395) (873) SE 20 38 27 7 1 Unit 93-3 879 817 351 820 95-1 738 686 321 810 05-7 551 371 357 83 96-2b 536 560 315 815 96-7 582 605 852 886 96-11 696 663 811 509 97-3b 689 638 339 815 98- 5 701 783 333 886 100- 5b 582 708 887 815 101 1b 526 782 321 368 Av. (609) (617) (365) (881) s 30 82 16 12 — — "Iva...“— aSigma urease tyte II powder. bOne-half of p138 in each treatment were fed rations containing urea. See Table 9 for treatments. . 1’3t5 . APPENDIX TABLE 11 TRIAL II. SERUM ANTIUREASE ACTIVITY IN IMMUNIZATION STUDY (unitalml. x 103)8 AA Pig Time. days No. In 29 8] 77_g7" Control 93-7b 38 -27 0 -3 98-7 -35 -27 -28 -2 98-9b -1 -27 35 28 95-3b 28 18 -28 18 96-3b 28 -81 33 -13 96-6 7 1 -13 -25 96-2 55 10 22 -5 97- 28 0 -7 32 100-1b 28 -82 16 -19 101-6 ~29 7 8 18 Av. (18) (-13) (8) (2) S 9 7 7 0.1 Unit 93-1 158 308 317 288 98.8b 193 196 239 165 95-8b 210 207 182 193 96-1b 221 178 198 176 96-9 195 308 205 195 97-2b 197 290 237 86 98-3 206 310 257 153 99-3 188 287 236 79 loo-8b 206 187 202 77 101-7 107 212 191 83 Av. (188)c (28g)d (222)dd (181,d SE 11 1 15 21 1 Unit 93-3b 267 258 109 115 95-1 51 270 179 79 95-7 197 177 136 138 96-2b 179 207 115 53 96-7 99 187 115 51 96-11 85 170 113 59 97-3 163 205 162 188 98-5b 20 158 261 57 100-5b 251 217 188 93 101-1 177 ~ 200 c 210 18 6 Av. (185)“ (208) (158)c (81) SE 27 12 16 13 aSigma urease type II powder. ane-half of pigs in eacn treatment were ”ed rations containing urea. See 8able 9 for treatments. cSignificantly greater than the least treatment mean (P-i 0.01). dSignificantly " " " other two treatment means (P #:0.05); dd(P < 0.01). .Amo.omi my mcome pcmeummnp 66636 66606 cunp umpmwnw h6pcwo6w6cm6mo .»Hm>6poommon.am>ma 6069 can 60660; 660>66 nous no>o .0076; 6:.m.©m-66 ”m.m.>6-mm “mm.6.mm-6: “60>06 60699 .6 .novaom H6 0 p 66606: new6mn 06.0 66.0 66.0 m6.0 mm 66.0 66.0 66.0 66.0 66 666.66 6666.66 666.66 666.66 .>6 666.66 666.66 606.66 606.66 .>6 06.6 66.6 66.6 m6.6 6-66 mm.6 00.6 66.6 ,m.6 6-606 66.6 66.6 .6 64.6 66-66 60.6 66.6 m6.6 6.6 6-006 06.6 66.6 6.6 00.6 6-66 66.6 mm.6 6.6 m6.6 m-66 66.6 m6.6 66.6 6.6 6-66 66.6 m6.6 66.6 6m.6 6-66 66.6 66.6 66.6 6.6 m-m6 06.6 00.6 66.6 6 .6 6-60 6669 6 66.0 66.0 66.0 06.0 mm 66.0 66.0 66.0 66.0 66 666.66 666.66 666.66 666.66 .>< 666.66 66m.6v 666.66 666.66 .>< 06.6 66.0 66.6 66.6 6-606 66.6 66.6 66.6 66.6 6-006 66.6 66.6 66.6 66.6 m-66 66.6 m6.6 m6.6 66.6 6-66 66.6 66.6 66.6 66.6 6-66 66.6 mm.6 m6.6 66.6 6-66 . 06.6 00.6 66.6 60.6 6-66 66.6 66.6 66.6 66.6 6-66 MM m6.6 66.6 66.6 M6.6 6-66 66.6 66.0 06.6 66.6 6-66 . 6606 6.0 06.0 60.0 66.0 06.0 mm 66.0 66.0 66.0 66.0 66 666.66 6666.66 666.66 666.66 .>« 666.66 666.66 666.66 666.66 .>< 66.6 66.6 60.6 66.6 6-006 06.6 00.6 66.6 66.6 6-606 66.6 66.6 66.6 M6.6 6-66 66.6 06.6 00.6 66.6 6-66 66.6 66.6 m6.6 66.6 6-66 66.6 60.6 66.6 6m.6 m-m6 66.6 66.6 66.6 66.6 m-66 66.6 mm.6 66.6 66.6 6-66 66.6 66.6 66.6 66.6 6-66 60.6 66.6 66.6 m6.6 6-m6 HOthOO .76 -6 -6 66-m67 m6-66 .02 66-66 .77mmuM6 66-m67-7, m6uM6 77 .0z 7 6 no 66568 7 .M6m 77 6666 .0868 mwm 7,117 6onpaoo - . 7 6663 II. 71 169E066 6626266666766 266967666 6860 may 60 am<6 666606 09 «mm: 666 6666 6262060 666620226202 az< 666623226 60 26<0 6666a moemm>< .66 64666 NH mqm<9 xHszmm< 1 111 ll! - 107 - APPENDIX TABLE 13 TRIAL II. PLASMA UREA N LEVELS OF IMMUNIZED AND NONIMMUNIZED GROWING PIGS FED UREA TO SUPPLY PART OF THE CRUDE PROTEIN REQUIREMENTS __ng.[l00 mlilé ___ _‘ b Urea . Control Pig Time, days Pig Timel_day3 No. .63-77 77189 No. 63177 77-8§_' Control 93-7 28.7 27.2 98-7 16.8 18.6 98-9 20.9 30.0 96-3 13.7 16.2 95-3 17.1 26.9 96-8 13.9 12.9 96-6 21.9 22.1 97-6 20. 21.7 101-6 28.5 36.0 100-1 18.9 15.0 Av. (21.8)c (28.8)cc Av. (15.8) (16.9) SE 1.8 2.3 SE 1.2 1.5 9;]: Unit 95-8 17.7 23.9 93-1 15.8 18.1 96-1 16.0 20.9 98-8 16.1 21.1 96-9 20.2 27.2 97-2 20.2 18.1 \8-3 21.9 30.6 99-3 20.8 21.3 100-8 22.3 28.0 101-7 11.1 9.5 Av. (19.6) (26.1)cc Av. (16.8) (16.8) SE 1.2 1.7 SE 1.8 2.2 1 Unit 95-1 22.5 28.2 93-3 23.8 29.2 96-2 12.8 30.1 95-7 26.8 28.0 97-3 13.8 20.9 96-7 19.2 20.5 100-5 31.5 35.8 96-11 18.5 17.5 101-1 18.8 13.2 98-5 23.2 21.8 Av. (19.3)d (25.6)cc Av. (22.2)cc (23.7.)c SE 3.8 3.9 SE 1.8 2.2 aSigma urease type II powder. burea levels: 63-77,2.5 and 77-89,3.8; period and urea level, respectively. Blood samples were taken at the end of the period. CSignificantly larger than the least two treatment means (P _0.0S); CC(P <0.01). dSignificantly H H N N treatment mean (P-< 0.05). - 108 - APPENDIX TABLE 18 TRIAL II. AMMONIA N LEVELS OF IMMUNIZED AND NONIMMUNIZED GROWING PIGS FED UREA TO SUPPLY PART OF THE CRUDE PROTEIN REQUIREMENT figch/IOO m1.)§ b Urea Content Vpig Time, days wfiié Time, days “0' I =§3'71 Z7-82;_ No. 63-77 17-89 Control 93-7 815 338 98-7 862 862 98"? 368 393 96-3 509 892 95-3 391 827 96-8 801 806 96‘s 3““ 201 97-6 377 815 101-6 862 380 100-1 862 878 Av. (395) (388) Av. (882) (850)CC 0.1 Unit 96'1 391 551 98-8 519 568 96-9 886 573 97-2 862 508 98‘3 “75 538 99-3 838 513 lOO-h 867 823 101-7 530 56“ AV' (858)e (Slh)d Av. (893)d (5u0)dd SE 17 27 SE 26 13 1 Unit 95‘1 810 385 93-3 820 538 96-2 815 385 95-7 886 879 97-3 815 329 96-7 886 598 100-5 1815 363 96-11 509 5914 101-1 368 880 98-5 886 587 AV- (805) (380) Av. (877)c (550)dd SE 9 18 SE 15 21 -6- _a..- a..." . _- .__, aSigma urease type II powder. bUrea levels: 03-77,2.5 and 77-89,3.8; period and urea level, respectively. Blood samples were taken at the end of the period. CSignificantly larger than the least two treatment means (P < 0.05);CC(P a 0.01). Significantly larger than the least three treatment means (P«<0.05);dd(P-< 0.01). eSignificantly larger than the least treatment mean (P7< 0.05). -109... APPENDIX TABLE 15 TRIAL III. SEX, INITIAL-WEIGHT, FINAL-WEIGHT,.AND AVERAGE DAILY GAIN 0F GROWING PIGS I! IMMUNIZATION STUDE: Pig Age Initial Final Time,3ggl§ J_ 170. Sex sags wt ugh . wt.,ib. 0-28 28-56 0-56 56-73" QQBEEEL 1-8b r 52 26 109 1.18 1.25 1.21 0.88 2-2b M 52 32 110 0.96 1.32 1.18 0.82 8-2 M 89 35 187 1.36 1.60 1.88 1.71 8-7b r 89 31 129 1.32 1.68 1.88 0.88 11-3b M 88 27 128 1.11 1.83 1.27 1.76 56-3 M 52 33 128 1.11 1.50 1.30 1.29 57-8 M 51 27 129 1.21 1.53 1.38 1.87 57-6 7 51 38 182 1.36 1.83 1.39 1.76 59-8b M 89 30 185 1.39 1.75 1.57 1.59 89-2 2 50 38 182 1.29 1.59 1.39 1.76 Av. (31) (1.23) 1.50) (1.36) (1.39 SE 0.08 0.05 0.08 0.13 0.5 t 1-5 F 52 38 129 1.32 1.32 1.32 1.28 8-8 p 89 33 152 1.50 1.68 1.59 1.76 8-5b r 89 30 128 1.83 1.53 1.88 0.88 6-1b M 88 26 118 1.36 1.39 1.38 0.88 11-1b M 88 32 128 1.39 1.32 1.36 0.98 11-8 M 88 32 137 1.32 1.50 1.81 1.53 56-1b M 52 31 128 1.18 1.60 1.39 1.00 57-2b M. 51 35 152 1.57 1.75 1.66 1.81 57-5 M 51 27 c 1.08 0 59-7 2 89 27 129 1.18 1.83 1.30 1.71 Av. (31) 1.33)‘1 (1.51) (1.83) (1.26) SE 0.05 0.05 0.08 0.12 Uhit 3-2b M 51 33 113 1.18 1.39 1.27 0.52 8-6b r 89 38 138 1.32 1.29 1.30 1.59 5-7 F 88 28 85 0.86 1.10 0.98 0.12 11-2 M 88 28 136 1.29 1.50 1.39 1.76 11-5b N. 88 26 110 1.25 1.36 1.30 0.65 56-2b M. 52 35 127 1.18 1.36 1.27 1.23 57-1 M 51 30 137 1.25 1.57 1.81 1.65 59-10 F 89 30 126 0.86 1.86 1.16 1.82 89-8b r 50 38 180 1.25 1.82 1.58 1.18 Av. (31) (1.15) (1.86) (1.31) (1.25) SE 0.05 0.07 0.05 0.20 aJ'ackbean urease prepared by the author. bOne-half of the pigs in each group were fed a ration with 3.8 percent urea added form 56 to 73 days. cPig died after 39 days; autOpsy showed stomach ulcer. dSignificantly greater than the least treatment mean (P-<0.05). TRIAL III. PLASMA UREA N LEVELS IN IMMUNIZATION STUDY (ngZIQO m1.)9 - 110 APPENDIX TABLE 16 v Pig Timei_d§ys _=_ No. 28 56 13? Control 1.8b 10.9 11.8 25.6 2—2b 15.3 16.6 36.5 8-2 18.7 21.8 19.7 8-7b 11.3 16.1 26.7 11-3 11.2 18.2 20.5 56-3 13.6 20.2 36.0 57-8 15.0 20.2 18.3 57-6 19.0 28.8 23.5 59-8 16.1 19.9 33.8 89-2 12.3 15.7 12.8 Av. (13.9) (18.8) (25.3) SE 0.8 1.2 2.5 0.5 Unit 1-5 13.7 13.0 13.0 8-8 19.2 23.6 20.8 8.5b 17.8 18.3 26.7 6-1b 16.2 12.8 30.0 11-1b 18.5 18.6 27.9 11-8 16.2 22.8 19.5 56-1b 18.9 19.7 30.8 57-2 12.3 15-3 37.0 57-5 11.9 d d 59-7 13.5 16.8 16.9 Av. (15.0) (17.8) (28.7) SE 0.7 1.1 2.5 5 Unit 3-2b 12.5 16.1 15.7 8-6 18.0 11.9 15.1 5-7b 16.3 16.8 17.0 11-2 12.7 21.8 19.8 11.5b 18.7 23.9 28.7 56-2b 13.7 19.5 28.1 57-1 11.2 18.1 12.3 57-3 12.0 16.3 15.5 59-10 10.8 16.9 16.5 89-8b 18.7 22.0 29.5 Av. (13.2) (17.9) (19.8) SE 0.6 1.2 1.9 aJ'ackbean urease prepared by the author. bOne-half of the pigs in each group were fed a ration with 3.8 percent urea added from 56-73 days. cPigs in each treatment were group fed. dPig died 39 days; autOpsy showed stomach ulcer. - lll - APPENDIX TABLE 17 781.8141; _a_IASMA @91qu 1 nevus IN WJIYATIOEIQDY mengOO 1:11.)“ ##- Pig A4_ Time. days ‘_____ N921 21.1 25 _1 _ 512 __ __ 71b 1 Control 1.8b 772 629 882 E-2b 623 £08 825 -2 7 5 50 8 S 8-7b 535 752 535 lé-3b 6&7 672 8:8 5 -3 7 7 730 5 3 57-8 716 885 582 57-6b 625 699 “97 59-8 800 881 865 89-2 627 531 889 Av. (695) (710)° (888)“3 SE 27 32 18 912.2222 1-5 692 557 818 8-8 678 553 818 8-5b 782 628 395 8 22.: “1:6 11-1 3 9 11-8b 655 553 889 56-1 730 577 525 572‘) 337 536 382 57-5 13 d 59-7 702 637 502 Av. (7:5) (622) (32:) SE 0 ’ 5 Unit 3-2b 698 752 828 8-6 678 713 818 s--7ID 789 881 535 11-2 730 787 535 82:: 22; $2: 22: 57-1 75“ 890 535 57-3 698 752 558 59-10 716 575 886 89-8b 656 659 395 Av. (688) (712)e (866)e SE 17 38 21 aJ'ackbean urease prepared by the author. b0ne-ha1f of the pigs in each group were fed a ration.w1th 3.8 percent urea added from S6 to 73 days. cPigs in each treatment were group fed. dDied 39 days; autopsy showed stomach ulcer. eSighificantly greater than the least treatment mean (P<:0.05); ee(P‘10.0l). - 1-4.? - APPENDIX TABLE 18 TRIAL III. SERUM ANTIUREASE ACTIVITY IN IMMUNIZATION STUDY ALunita/ml. x_10§lf AA“ _;‘ _A —_ - P18 _ _¢_3 Time; days E9;1_1 .1:§§-ne_.___..__ 2§,__. _,,=g;=:73§ Contro1 1.8b -11 -28 1 2-2b -26 19 3 8-2 37 53 0 8.7b 28 -19 -10 11-3b -83 2 -11 56-3 26 -25 25 57-8 -12 8 13 57-6 -22 53 2 g9-8b BE ~30 0 9-2 -23 -15 2. 9 :32 a? 0.5 Unit 1-5 88 29 60 8-8 81 87 20 8-5b 123 57 50 6-1: 118 87 86 11-1 88 75 18 11-8 85 21 39 56-1b 25 6 29 57-2b 9o 58 86 57-5 106 - - 59-7 101 82 28 Av. (98)9 (87)e (32)e SE 9 5 Unit 3-2b 117 26 29 8-6 131 60 80 1:2: .. . :2 11-5b 89 39 22 56-2 83 93 50 57-1 120 23 15 57‘3 10h 87 38 397% “a $6 5; 9- - 0 3 Av. (88)e (67)° (35)e SE 18 13 8 WW wvv—f aJ'ack‘hean urease prepared by the author. b0ne-ha1r of the pi s in each group were fed a ration with 3.8 percent urea added from %6 to 73 days. cPigs in each treatment were group fed. dnied 39 days; autopsy showed stomach ulcer. eSignificantly greater than the least treatment mean (P<:0.01). .Aao.0t.mv mecca phenomena neon . a e . aaoonoaueamamo .AH0.0V.mv memos anoeeeona tonne . c . e aaoeaoauanmnme .Am0.0v.mv ones phenomena amend one can» nooaohm aaaaaoaafiamamo .Amham m» o» mmv ammo NH you mcoauau any: new mwam .vnuaauvmxo on» no one 059 pa aoxav endgame ooonn .uonnaa on» an ooudnohn mucous octagonna em >.H 00.0 am am 0.A 00.0 mm 2.0 3.3 .33 .2 33 .033 8.0 .2 - - - -. mom 0.em ae.a ~-em mom 0.0a H~.H p.0m mum 0.0m 00.a 8-0m mm: «.ma mm.a :-HH 03m 0.e~ e0.0 H-HH 4H: 0.0m m~.H 4-: we: 0.0m 00.0 H-m . ea: 0.ma :m.a m-« mam p.00 00.0 m.: 0. $84.... . em ».H “0.0 an em m.m ma.0 mm A81; 80$ $00.: .2 85 88.5 80.3 .3. owe A.ma we.» m-0m awe A.mm 0m.a e-0n >0: m.m~ 0~.H m.em men 0.0m mm.a n-0m «mm m.ma ~e.a anew «mm ~.mm 00.0 >-: 0H: n.0m me.a m-aa m0: m.mm «0.0 «-0 mm: A.ma He.a «-4 we: m.mm 00.0 e-H dogwooo .As 00H\Auoa .As 00H\8ma .hH Amenml. .02 .Ha 00Hw.ucs .As 00H\.um. .aa .oaam. .02 z nanoaa< 2 one: .me .>< mwmi z oHeoaa< a can: .00 .>< man Honpooo non: no gag ZHQHOE @955 E ho 85E Emma on. g EOE :.m E 8Hm ENHZDEfizoz Q24 3,525” .8 “Hg 2 < Ammev A0.mmv Aee.0v .>< . we: n.0H «0.A 0H-0m «mm «.00 0H.A 8-0m 0mm «.ma 00.0 m-~m mm: A.mm m~.~ 0-0m mmm m.~a m0.a H->m 00m e.em no.0 «-AH mmm m.ma w».a Nuaa mmm o.~a Na.o pnm ma: A.ma mm.a m-: am: >.ma mm.o m-m one: w .83 00H\muna .As oomwmma .oa780mmmr. .02 .As 00Hw.uca .Ha 00H\mum7 .oH .mwemll. .02 a eaooaa< .72 ammo .00 .><_ mam z oaaoss< a can: .ao .>< mam H OhHQOO “Us .7 .As mazmxumumamm zmwaomm mommw are no amen mammam 09 «um: azmommm e.mns 00m noun amqumxtzoz 02¢.0MNH2022H a0 mqmsma z < .HHH qeama AQMDZHBZOUV ma mqm<fi Nanmmm< - 115 - APPENDIX TABLE 20 TRIAL III. UREASE ACTIVITY AND AMMONIA N LEVELS OF IM'ESTINAL ,__U§E§ILQNS PLU§_COUI§ETS FROM PIG$_IN_IMMUNIZATION STUD! “b Pig urease activity Ammonia N No. _r g___ unitalgm. DM x 103 ‘_3 mcg.[gg. DM 99211291 1.8c 38.7 258 8-2 28.1 83 11-3c 53.8 310 39.8 30.2 132 9-2 3102 1 AV. (3307) (182) as 8.3 36 932.9223 8.8c 27.8 187 8-5 37.5 188 11-1° 28.8 113 11.8 8.7 128 57-2° 38.9 200 Q? (22.?) (131;) unit 3-2° 8.8 135 8-6 16.9 191 56-2c 19.0 129 59-10 15.3 90 89.8c 26.3 380 Av. (16.5)dl (172) SE 3.3 36 aJ'ackbean urease prepared by the author. bPigs slaughtered at the end of experiment, 73 days. In- testinal sections plus contents were taken from the lower end of the i cum. These pigs received rations containing 3.8 percent urea from 56 tod73 days. Immunized pigs had significantly (P<:0.05) less urease activity than control pigs. - 116 - APPENDIX TABLE 21 TRIAL I. PLASMA AND URINE UREA N AND AMMONIA N LEVELS IN TOXICITY STUDY ab 1...::===============.e===e2:: Plasma Urea N Plasma Ammonia N mg./100 m1. n3.;100 ml. - Time Control 100 Unit Control 100 Unit -21 18.8 18.1 368 822 0 12.8 10.3 658 675 15 min. 11.5 8.9 61% 720 85 " 11.9 5.7 658 1317 75 " 11.5 3.0 856 1255 8 hr. 10.2 2.2 882 1519 8 " 9.2 0.5 992 3833 9 " c 2.8 c 3558 10 " c 2.9 c 2989 Av. (11.6) (5.6)d (608) (1760)dd Urine Urea N Urine Ammonia N Ulfi'/hr’ ”1&1!Ihra Pre Post Pre Post . injectione injectione Change injection injection Change Control 102 67 -35 5.6 1.5 ~h.1 100 Unit 155 102 -53 3.1 8.8 1.7 aJ’ackbean urease prepared by the author. bTwo pigs: 69-10, control and 68-5, 100 units; weighed 30 and 35 pounds, respect=vely. Pigs were fasted during entire trial. CSample not taken. dSignificantly different from control (P <20.05);dd(P .;o.01). eTwenty hour pro-injection and eight hour post-injection collection period. - 117 - APPENDIX TABLE 22 TRIAL II._~PLASMA UREA AND AMMONIA N LEVELS IN TOXICITY STUDY ab Pig =: TimeA hours ~_¥ Urea ngmg./100 ml. Control 87—5 18.8 9.3 9.0 10.3 9.5 11.8 13.0 78-6 16.5 11.2 10.2 9.8 9.7 10-9 12-2 A (15.6) (10.2) (9.6)c (9.8)d (9.1)d (11.3)c (12.6)c SE 0.9 1.0 0.6 0.5 0.1 0.5 0.8 50 Units 87-2 11.6 9.8 8.8 5.8 5.1 6.7 10.9 78-7 13.0 2.8 9.0 5.6 5.6 9.8 12.8 Av. (12.3) (10.9) (8.7) (5.5) (5.2) (8.0) (11.6) SE 0.7 1.5 0.3 0.1 0.3 1.8 0.8 177112: 78-8 18.8 9.2 5.2 3.8 2.6 3.8 8.7 97-8 9.8 9.6 7.8 5.1 8.5 5.8 8.8 Av. (12.1) (9.8) (6.3) (8.2) (3.6) (8.8) (6.5) $3 2.3 0.2 1.1 1.0 1.0 1.0 1.9 Ammonia N, mcg,/1OO ml. Control 87-5 566 615 566 608 599 1176 750 78-6 593 788 539 608 917 1133 888 Av- (580) (680) (552) (608) (758) (1158) (917) SE 18 68 18 0 159 21 67 50 Unit 87-2 577 593 712 1213 1278 1726 1197 79-7 653 725 309 1537 1952 1753 1731 Av. (615) (660) (760) (1375)c (1615) (1739) (1868) SE 38 67 89 162 337 18 267 100 Unit 78-8 585 631 1321 1568 2217 2589 8660 87-8 626 687 512 1172 2118 2001 1327 Av. (585) (639) (916) (1368)° (2165)° (2295)c (2993) SE 81 8 806 196 51 298 1665 aJ'ackbean urease prepared by the author. bPigs 78-7, 78-8 and “7-3 died aftc~ +:.e iriecticn. 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